RESUMO
Remote ischemic preconditioning (RIPC) exerts a protective role on myocardial ischemia/reperfusion (I/R) injury by the release of various humoral factors. Lactate is a common metabolite in ischemic tissues. Nevertheless, little is known about the role lactate plays in myocardial I/R injury and its underlying mechanism. This investigation revealed that RIPC elevated the level of lactate in blood and myocardium. Furthermore, AZD3965, a selective monocarboxylate transporter 1 inhibitor, and 2-deoxy-d-glucose, a glycolysis inhibitor, mitigated the effects of RIPC-induced elevated lactate in the myocardium and prevented RIPC against myocardial I/R injury. In an in vitro hypoxia/reoxygenation model, lactate markedly mitigated hypoxia/reoxygenation-induced cell damage in H9c2 cells. Further studies suggested that lactate contributed to RIPC, rescuing I/R-induced autophagy deficiency by promoting transcription factor EB (TFEB) translocation to the nucleus through activating the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway without influencing the phosphatidylinositol 3-kinase-Akt pathway, thus reducing cardiomyocyte damage. Interestingly, lactate up-regulated the mRNA and protein expression of connexin 43 (CX43) by facilitating the binding of TFEB to CX43 promoter in the myocardium. Functionally, silencing of TFEB attenuated the protective effect of lactate on cell damage, which was reversed by overexpression of CX43. Further mechanistic studies suggested that lactate facilitated CX43-regulated autophagy via the AMPK-mTOR-TFEB signaling pathway. Collectively, this research demonstrates that RIPC protects against myocardial I/R injury through lactate-mediated myocardial autophagy via the AMPK-mTOR-TFEB-CX43 axis.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Conexina 43 , Traumatismo por Reperfusão Miocárdica , Serina-Treonina Quinases TOR , Animais , Masculino , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Precondicionamento Isquêmico/métodos , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Glioblastoma multiform (GBM) is a highly aggressive primary brain tumor. Exosomes derived from glioma cells under a hypoxic microenvironment play an important role in tumor biology including metastasis, angiogenesis and chemoresistance. However, the underlying mechanisms remain to be elucidated. In this study, we aimed to explore the role of connexin 43 on exosomal uptake and angiogenesis in glioma under hypoxia. U251 cells were exposed to 3% oxygen to achieve hypoxia, and the expression levels of HIF-1α and Cx43, involved in the colony formation and proliferation of cells were assessed. Exosomes were isolated by differential velocity centrifugation from U251 cells under normoxia and hypoxia (Nor-Exos and Hypo-Exos), respectively. Immunofluorescence staining, along with assays for CCK-8, tube formation and wound healing along with a transwell assay were conducted to profile exosomal uptake, proliferation, tube formation, migration and invasion of HUVECs, respectively. Our results revealed that Hypoxia significantly up-regulated the expression of HIF-1α in U251 cells as well as promoting proliferation and colony number. Hypoxia also increased the level of Cx43 in U251 cells and in the exosomes secreted. The uptake of Dio-stained Hypo-Exos by HUVECs was greater than that of Nor-Exos, and inhibition of Cx43 by 37,43gap27 or lenti-Cx43-shRNA efficiently prevented the uptake of Hypo-Exos by recipient endothelial cells. In addition, the proliferation and total loops of HUVECs were remarkably increased at 24 h, 48 h, and 10 h after Hypo-Exos, respectively. Notably, 37,43gap27, a specific Cx-mimetic peptide blocker of Cx37 and Cx43, efficiently alleviated Hypo-Exos-induced proliferation and tube formation by HUVECs. Finally, 37,43gap27 also significantly attenuated Hypo-Exos-induced migration and invasion of HUVECs. These findings demonstrate that exosomal Cx43 contributes to glioma angiogenesis mediated by Hypo-Exos, and suggests that exosomal Cx43 might serve as a potential therapeutic target for glioblastoma.
Assuntos
Exossomos , Glioblastoma , MicroRNAs , Neovascularização Patológica , Hipóxia Celular , Linhagem Celular Tumoral , Conexina 43/genética , Conexina 43/metabolismo , Células Endoteliais/metabolismo , Exossomos/metabolismo , Glioblastoma/genética , Humanos , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Microambiente TumoralRESUMO
The proliferation of pulmonary artery smooth muscle cells (PASMCs) is an important cause of pulmonary vascular remodelling in hypoxia-induced pulmonary hypertension (HPH). However, its underlying mechanism has not been well elucidated. Connexin 43 (Cx43) plays crucial roles in vascular smooth muscle cell proliferation in various cardiovascular diseases. Here, the male Sprague-Dawley (SD) rats were exposed to hypoxia (10% O2 ) for 21 days to induce rat HPH model. PASMCs were treated with CoCl2 (200 µM) for 24 h to establish the HPH cell model. It was found that hypoxia up-regulated the expression of Cx43 and phosphorylation of Cx43 at Ser 368 in rat pulmonary arteries and PASMCs, and stimulated the proliferation and migration of PASMCs. HIF-1α inhibitor echinomycin attenuated the CoCl2 -induced Cx43 expression and phosphorylation of Cx43 at Ser 368 in PASMCs. The interaction between HIF-1α and Cx43 promotor was also identified using chromatin immunoprecipitation assay. Moreover, Cx43 specific blocker (37,43 Gap27) or knockdown of Cx43 efficiently alleviated the proliferation and migration of PASMCs under chemically induced hypoxia. Therefore, the results above suggest that HIF-1α, as an upstream regulator, promotes the expression of Cx43, and the HIF-1α/Cx43 axis regulates the proliferation and migration of PASMCs in HPH.
Assuntos
Conexina 43/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Conexina 43/agonistas , Conexina 43/genética , Hipóxia/genética , Hipóxia/metabolismo , Imuno-Histoquímica , Modelos Biológicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , RatosRESUMO
Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Gencitabina , Histona Acetiltransferases , Hialuronoglucosaminidase , Neoplasias Pancreáticas , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Gencitabina/farmacologia , Gencitabina/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Hialuronoglucosaminidase/efeitos dos fármacos , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Histona Acetiltransferases/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/farmacocinética , Antígenos de NeoplasiasRESUMO
Long noncoding RNAs (lncRNAs) are related to the development of atherosclerosis (AS). However, the role of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in tumor necrosis factorα (TNFα)induced rat aortic endothelial cell (RAOEC) pyroptosis, as well as the underlying mechanisms, remain unclear. RAOEC morphology was assessed using an inverted microscope. The mRNA and/or protein expression levels of MALAT1, microRNA(miR)30c5p and connexin 43 (Cx43) were assessed using reverse transcriptionquantitative PCR (RTqPCR) and/or western blotting, respectively. The relationships among these molecules were validated by dualluciferase reporter assays. Biological functions, such as LDH release, pyroptosisassociated protein levels and the proportion of PIpositive cells, were evaluated using a LDH assay kit, western blotting and Hoechst 33342/PI staining, respectively. The present study demonstrated that compared with the control group, the mRNA expression levels of MALAT1 and protein expression levels of Cx43 were significantly upregulated, whereas miR30c5p mRNA expressions levels were significantly decreased in TNFαtreated RAOEC pyroptosis. Knockdown of MALAT1 or Cx43 significantly attenuated the increase in LDH release, pyroptosisassociated protein expression and PIpositive cell numbers among RAOEC treated using TNFα, whereas an miR30c5p mimic exerted the opposite effect. Furthermore, miR30c5p was demonstrated to be a negative regulator of MALAT1 and could also target Cx43. Finally, cotransfection with siMALAT1 and miR30c5p inhibitor could attenuate the protective effect of MALAT1 knockdown against TNFαmediated RAOEC pyroptosis by upregulation of Cx43 expression. In conclusion, MALAT1 might serve an important role in TNFαmediated RAOEC pyroptosis by regulating the miR30c5p/Cx43 axis, which would provide a potential novel diagnostic and therapeutic target for AS.
Assuntos
MicroRNAs , RNA Longo não Codificante , Ratos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Piroptose/genética , Conexina 43/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Células Endoteliais/metabolismo , RNA MensageiroRESUMO
Corneal alkali burn, one of the most serious ophthalmic emergencies, is difficult to be cured by conservative treatments. It is well known that oxidative stress, inflammation and neovascularization are the main causes of corneal damage after alkali burn, but its underlying mechanism remains to be elucidated. Here, we reported that the expression and phosphorylation (Ser616) of mitochondrial fission protein Drp1 were up-regulated at day 3 after alkali burn, while mitochondrial fusion protein Mfn2 was down-regulated. The phosphorylation of ERK1/2 in corneas was increased at day 1, 3, 7 and peaked at day 3 after alkali burn. In human corneal epithelial cells (HCE-2), NaOH treatment induced mitochondrial fission, intracellular ROS production and mitochondrial membrane potential disruption, which was prevented by Drp1 inhibitor Mdivi-1. In corneas, Mdivi-1 or knockdown of Drp1 by Lenti-Drp1 shRNA attenuated alkali burn-induced ROS production and phosphorylation of IκBα and p65. In immunofluorescence staining, it was detected that Mdivi-1 also prevented NaOH-induced nuclear translocation of p65 in HCE-2 cells. Moreover, the expression of NADPH oxidase NOX2 and NOX4 in corneas peaked at day 7 after alkali burn. Mdivi-1, Lenti-Drp1 shRNA or the mitochondria-targeted antioxidant mito-TEMPO efficiently alleviated activation of NF-κB, expression of NOX2/4 and inflammatory cytokines including IL-6, IL-1ß and TNF-α in corneas after alkali burn. In pharmacological experiments, both Mdivi-1 and NADPH oxidases inhibitor Apocynin protected the corneas against alkali burn-induced neovascularization. Intriguingly, the combined administration of Mdivi-1 and Apocynin had a synergistic inhibitory effect on corneal neovascularization after alkali burn. Taken together, these results indicate that Drp1-dependent mitochondrial fission is involved in alkali burn-induced corneal injury through regulating oxidative stress, inflammatory responses and corneal neovascularization. This might provide a novel therapeutic target for corneal injury after alkali burn in the future.
Assuntos
Queimaduras Químicas , Lesões da Córnea , Dinâmica Mitocondrial , Animais , Queimaduras Químicas/tratamento farmacológico , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/tratamento farmacológico , Lesões da Córnea/genética , Dinaminas/genética , Humanos , Camundongos , MitocôndriasRESUMO
Alzheimer's disease (AD), one of the most common types of chronic neurodegenerative diseases, is pathologically characterized by the formation of amyloid ß (Aß) peptidecontaining plaques and neurofibrillary tangles. Among Aß peptides, Aß142 induces neuronal toxicity and neurodegeneration. In our previous studies, Cdk5 was found to regulate Aß142induced mitochondrial fission via the phosphorylation of dynaminrelated protein 1 (Drp1) at Ser579. However, whether blockage of Drp1 phosphorylation at Ser579 protects neurons against Aß142induced degeneration remains to be elucidated. Thus, the aim the present study was to examine the effect of mutant Drp1S579A on neurodegeneration and its underlying mechanism. First, the phosphorylationdefect (phosphodefect) mutant, LentiDrp1S579A was constructed. Phosphodefect Drp1S579A expression was detected in primary cultures of mouse cortical neurons infected with LentiDrp1S579A using western blotting and it was found to successfully attenuate the phosphorylation of endogenous Drp1 at Ser579. In primary neuronal cultures, the neuronal processes were evaluated under microscopy. Treatment with 10 µM Aß142 significantly decreased dendritic density and length, spine outgrowth and synapse number. As expected, infection of neurons with LentiDrp1S579A efficiently alleviated the inhibitory effect of Aß142 on neurite outgrowth and synapse density. In addition, infection with LentiDrp1S579A abolished the cleavage of caspase3 and apoptosis in neurons exposed to Aß142. Thus, the current data demonstrated that blockage of Drp1 phosphorylation at Ser579 may be an effective strategy to protect neurons against Aß142induced degeneration and apoptosis. These findings underline the therapeutic potential of targeting Drp1 in the treatment of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Apoptose/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas , Fragmentos de Peptídeos/farmacologia , FosforilaçãoRESUMO
Glioblastoma is the most common and aggressive brain tumor and it is characterized by a high mortality rate. Temozolomide (TMZ) is an effective chemotherapy drug for glioblastoma, but the resistance to TMZ has come to represent a major clinical problem, and its underlying mechanism has yet to be elucidated. In the present study, the role of exosomal connexin 43 (Cx43) in the resistance of glioma cells to TMZ and cell migration was investigated. First, higher expression levels of Cx43 were detected in TMZresistant U251 (U251r) cells compared with those in TMZsensitive (U251s) cells. Exosomes from U251s or U251r cells (sExo and rExo, respectively) were isolated. It was found that the expression of Cx43 in rExo was notably higher compared with that in sExo, whereas treatment with rExo increased the expression of Cx43 in U251s cells. Additionally, exosomes stained with dioctadecyloxacarbocyanine (Dio) were used to visualized exosome uptake by glioma cells. It was observed that the uptake of Diostained rExo in U251s cells was more prominent compared with that of Diostained sExo, while 37,43Gap27, a gap junction mimetic peptide directed against Cx43, alleviated the rExo uptake by cells. Moreover, rExo increased the IC50 of U251s to TMZ, colony formation and Bcl2 expression, but decreased Bax and cleaved caspase3 expression in U251s cells. 37,43Gap27 efficiently inhibited these effects of rExo on U251s cells. Finally, the results of the wound healing and Transwell assays revealed that rExo significantly enhanced the migration of U251s cells, whereas 37,43Gap27 significantly attenuated rExoinduced cell migration. Taken together, these results indicate the crucial role of exosomal Cx43 in chemotherapy resistance and migration of glioma cells, and suggest that Cx43 may hold promise as a therapeutic target for glioblastoma in the future.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Conexina 43/metabolismo , Glioma/tratamento farmacológico , Temozolomida/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Exossomos/metabolismo , Glioma/patologia , Humanos , Temozolomida/uso terapêuticoRESUMO
Cisplatin (DDP) is the first-line chemotherapeutic agent for ovarian cancer. However, the development of DDP resistance seriously influences the chemotherapeutic effect and prognosis of ovarian cancer. It was reported that DDP can directly impinge on the mitochondria and activate the intrinsic apoptotic pathway. Herein, the role of mitochondrial dynamics in DDP chemoresistance in human ovarian cancer SKOV3 cells was investigated. In DDP-resistant SKOV3/DDP cells, mitochondrial fission protein DRP1 was down-regulated, while mitochondrial fusion protein MFN2 was up-regulated. In accordance with the expression of DRP1 and MFN2, the average mitochondrial length was significantly increased in SKOV3/DDP cells. In DDP-sensitive parental SKOV3 cells, downregulation of DRP1 and upregulation of mitochondrial fusion proteins including MFN1,2 and OPA1 occurred at day 2~6 under cisplatin stress. Knockdown of DRP1 or overexpression of MFN2 promoted the resistance of SKOV3 cells to cisplatin. Intriguingly, weaker migration capability and lower ATP level were detected in SKOV3/DDP cells. Respective knockdown of DRP1 in parental SKOV3 cells or MFN2 in SKOV3/DDP cells using siRNA efficiently reversed mitochondrial dynamics, migration capability and ATP level. Moreover, MFN2 siRNA significantly aggravated the DDP-induced ROS production, mitochondrial membrane potential disruption, expression of pro-apoptotic protein BAX and Cleaved Caspase-3/9 in SKOV3/DDP cells. In contrast, DRP1 siRNA alleviated DDP-induced ROS production, mitochondrial membrane potential disruption, expression of pro-apoptotic protein BAX and Cleaved Caspase-3/9 in SKOV3 cells. Thus, these results indicate that mitochondrial dynamics mediated by DRP1 and MFN2 contributes to the development of DDP resistance in ovarian cancer cells, and will also provide a new strategy to prevent chemoresistance in ovarian cancer by targeting mitochondrial dynamics.
RESUMO
Alzheimer's disease (AD), with a typical pathological hallmark of amyloidbeta (Aß)containing plaques and neurofibrillary tangles, is one of the most common types of chronic neurodegenerative diseases. Aß oligomers serve a crucial role in the pathogenesis of AD, and lead to neuronal loss. However, the precise mechanism of Aß oligomers in AD remains to be elucidated. The present study demonstrated that 10 µM Aß42 activated the caspase signaling pathway, and induced significant apoptosis in primary cultured mouse cerebral cortical neurons. The results of reverse transcriptionquantitative polymerase chain reaction and western blotting demonstrated that Aß42 (10 µM) also significantly upregulated the transcription and expression of the mitochondrial fission protein dynaminrelated protein 1 (Drp1), and downregulated the transcription and expression of mitochondrial fusion proteins, including mitofusin 1/2 (Mfn1/2) and mitochondrial dynamin like GTPase (OPA1). Neurons were transfected with pDsRed2Mito for mitochondrial imaging, which revealed that 10 µM Aß42 induced mitochondrial fission in cortical neurons. In addition, 2',7'dichlorodihydrofluorescein diacetate and tetramethylrhodamine ethyl ester staining indicated that Aß42 increased the reactive oxygen species (ROS) level and reduced mitochondrial membrane potential in neurons. Inhibition of Drp1 activity by Mdivi1 efficiently prevented Aß42induced ROS production and disruption of mitochondrial membrane potential. Loss of mitochondrial membrane potential may activate PTENinduced putative kinase 1 (Pink1), the prominent sensor for mitochondrial damage, and trigger the process of mitophagy to remove the damaged mitochondria. In the present study, western blotting revealed that the levels of autophagy marker microtubuleassociated proteins 1A/1B light chain 3B (LC3B) and Pink1 were upregulated after Aß42 stimulation. In conclusion, these data indicated that Aß42 induces neuronal apoptosis by targeting mitochondria, including promotion of mitochondrial fission, disruption of mitochondrial membrane potential, increasing intracellular ROS level and activation of the process of mitophagy. Therefore, mitochondria may represent a potential therapeutic target for AD in the future.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apoptose , Mitocôndrias/metabolismo , Neurônios/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Mitofagia , Imagem Molecular , Neurônios/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondria are high dynamic organelles with frequent fission and fusion. Here, we found hypoxia stimulated Drp1 expression, mitochondrial fission and migration in metastatic MDA-MB231 cells, but not in non-metastatic MCF-7 cells. Inhibition of Drp1-dependent mitochondrial fission by Mdivi-1 or silencing Drp1 attenuated hypoxia-induced mitochondrial fission and migration in MDA-MB231 cells. On the other hand, cisplatin induced significant apoptosis and mitochondrial fission in MDA-MB231 cells, but not in MCF-7 cells. Mdivi-1 and silencing Drp1 also efficiently prevented cisplatin-induced MMP decrease, ROS production and apoptosis in MDA-MB231 cells. Our data suggest that Drp1-dependent mitochondrial fission not only regulates hypoxia-induced migration of breast cancer cells, but also facilitates its sensitivity to chemotherapeutic agents. Thus, targeting Drp1-dependent mitochondrial dynamics may provide a novel strategy to suppress breast cancer metastasis and improve the chemotherapeutic effect in the future.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Cisplatino/administração & dosagem , GTP Fosfo-Hidrolases/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/biossíntese , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dinaminas , Feminino , GTP Fosfo-Hidrolases/antagonistas & inibidores , Humanos , Células MCF-7 , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/antagonistas & inibidores , Quinazolinonas/administração & dosagemRESUMO
Glioblastoma is one of the most aggressive brain tumors with high morbidity and mortality. Hypoxia is often the common characteristic of tumor microenvironment, and hypoxia-inducible factor-1α (HIF-1α) is an essential factor regulating the migratory activity of cancer cells including glioblastoma. Recently, mitochondrial dynamics was found to be involved in the aggression of cancer cells. However, whether dynamin-related protein 1 (Drp1) contributes to the migration of human glioblastoma cells under hypoxia remains unknown. In the present study, hypoxia was found to upregulate the transcription and expression of Drp1, and stimulated mitochondrial fission in glioblastoma U251 cells. Inhibition of HIF-1α with echinomycin blocked hypoxiainduced expression of Drp1. Notably, Drp1 inhibitor Mdivi-1 efficiently attenuated hypoxia-induced mitochondrial fission and migration of U251 cells. In addition, three U251 stable cell lines expressing GFP, GFP-Drp1 and dominant negative GFP-Drp1K38A were established to examine the direct role of Drp1 in hypoxia-induced migration. MTT assay showed that there was no significant difference in proliferation of three cell lines. Compared with the GFP cell line, exogenously expressed GFP-Drp1-K38A inhibited hypoxia-induced migration of U251 cells, while stable expression of GFP-Drp1 enhanced the migration of U251 cells under hypoxia. Therefore, this study indicates the involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells, and suggests Drp1 to be a potential therapeutic target to suppress the aggression of glioblastoma in the future.