RESUMO
Objective. Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the CPE gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the CPE gene was studied by quantitative RT-PCR and normalized to ACTB. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of CPE gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the CPE gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of CPE gene in both types of used cells, but ERN1 inhibition enhanced this effect. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic CPE gene through protein kinase activity of ERN1 and that increased CPE gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.
Assuntos
Carboxipeptidase H , Estresse do Retículo Endoplasmático , Endorribonucleases , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Proteínas Serina-Treonina Quinases , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Carboxipeptidase H/metabolismo , Carboxipeptidase H/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Glucose/metabolismo , Técnicas de Silenciamento de Genes , Hipóxia Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Hypoxia occurs in 90% of solid tumors and is strongly associated with an increased propensity for metastasis. Hypoxia induces tumor progression largely through inducing HIF-mediated transcription, resulting in alterations to tumor cell metabolism, as well as increases in migration and invasion. Hypoxia also results in a myriad of changes to the tumor microenvironment (TME). While many studies have examined the immediate effects of hypoxia on tumor cells and the associated TME, far fewer have focused on the long-term consequences of transient reductions in oxygen. In this issue of Cancer Research, Iriondo and colleagues examined whether short-term exposure to hypoxia leads to a "hypoxic memory" in the context of breast cancer. The authors used established cell lines and circulating tumor cell lines to demonstrate that these cells harbor a hypoxic memory that sustains downregulation of IFN signaling and antigen presentation (AP) pathways that contribute to tumor progression via alterations to tumor cells and the TME. The authors further showed that cells that have experienced hypoxia maintain the reduction in IFN signaling in vivo and are more aggressive. They determined that the hypoxic memory and reduction of IFN signaling can be reversed with a histone deacetylase inhibitor, entinostat, providing a potential means to reverse hypoxia-induced suppression of IFN signaling. As suppression of IFN signaling has the potential to influence both tumor cells and the TME, the identification of a strategy to inhibit long-term suppression of IFN signaling downstream of hypoxia could prove to be an effective means to target tumor progression. See related article by Iriondo et al., p. 3141.
Assuntos
Neoplasias da Mama , Progressão da Doença , Transdução de Sinais , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Microambiente Tumoral , Interferons/metabolismo , Oxigênio/metabolismo , Hipóxia Celular , Animais , Linhagem Celular TumoralRESUMO
The effects of HIF1A knockdown by RNA interference on the histone H3K9 methylation in human umbilical cord mesenchymal stromal cells in vitro under conditions of 24-h exposure to hypoxia (1% O2) were studied. Evaluation of transcriptional activity of genes involved in the regulation of H3K9 methylation (KDM3A, KDM4A, and EHMT2) and the cytofluorimetric analysis of the expression of the corresponding antigens and H3K9 methylation level demonstrated a pronounced stimulating effect of hypoxic exposure. Moreover, the expression of KDM4A and EHMT2 was regulated by HIF1A-mediated mechanism, unlike KDM3A; the level of the corresponding proteins depended on HIF1A. In addition, the HIF-1-dependent regulation of KDM3A, KDM4A, and EHMT2/G9a, and directly the H3K9 methylation level in mesenchymal stromal cells also took place under normoxia conditions.
Assuntos
Hipóxia Celular , Histonas , Subunidade alfa do Fator 1 Induzível por Hipóxia , Histona Desmetilases com o Domínio Jumonji , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Humanos , Histonas/metabolismo , Histonas/genética , Metilação , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia Celular/genética , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Interferência de RNA , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Regulação da Expressão GênicaRESUMO
Solid tumours are complex and heterogeneous systems, which exist in a dynamic biophysical microenvironment. Conventional cancer research methods have long relied on two-dimensional (2D) static cultures which neglect the dynamic, three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform, which allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion in IFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.
Assuntos
Líquido Extracelular , Dispositivos Lab-On-A-Chip , Esferoides Celulares , Transcriptoma , Microambiente Tumoral , Humanos , Esferoides Celulares/metabolismo , Líquido Extracelular/metabolismo , Perfusão , Linhagem Celular Tumoral , Hipóxia Celular , Neoplasias/metabolismo , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Rationale: Glioma stem cells (GSCs) have emerged as pivotal drivers of tumor malignancy, sustained by various microenvironmental factors, including immune molecules and hypoxia. In our previous study, we elucidated the significant role of transforming growth factor beta-induced protein (TGFBI), a protein secreted by M2-like tumor-associated macrophages, in promoting the malignant behavior of glioblastoma (GBM) under normoxic conditions. Building upon these findings, the objective of this study was to comprehensively explore the crucial role and underlying mechanisms of autocrine TGFBI in GSCs under hypoxic conditions. Methods: We quantified TGFBI expression in glioma specimens and datasets. In vitro and in vivo assays were employed to investigate the effects of TGFBI on sustaining self-renewal and tumorigenesis of GSCs under hypoxia. RNA-seq and LC-MS/MS were conducted to explore TGFBI signaling mechanisms. Results: TGFBI is preferentially expressed in GSCs under hypoxic conditions. Targeting TGFBI impair GSCs self-renewal and tumorigenesis. Mechanistically, TGFBI was upregulated by HIF1α in GSCs and predominantly activates the AKT-c-MYC signaling pathway in GSCs by stabilizing the EphA2 protein through preventing its degradation. Conclusion: TGFBI plays a crucial role in maintaining the stem cell properties of GSCs in the hypoxic microenvironment. Targeting the TGFBI/EphA2 axis emerges as a promising and innovative strategy for GBM treatment, with the potential to improve the clinical outcomes of patients.
Assuntos
Glioma , Células-Tronco Neoplásicas , Receptor EphA2 , Fator de Crescimento Transformador beta , Microambiente Tumoral , Células-Tronco Neoplásicas/metabolismo , Humanos , Receptor EphA2/metabolismo , Animais , Glioma/metabolismo , Glioma/patologia , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular Tumoral , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Transdução de Sinais , Proteínas da Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia Celular , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Hipóxia/metabolismo , Carcinogênese/metabolismoRESUMO
The hypoxic tumor microenvironment significantly impacts cellular behavior and intercellular communication, with extracellular vesicles (EVs) playing a crucial role in promoting angiogenesis, metastasis, and host immunosuppression, and presumed cancer progression and metastasis are closely associated with the aberrant surface N-glycan expression in EVs. We hypothesize that hypoxic tumors synthesize specific hypoxia-induced N-glycans in response to or as a consequence of hypoxia. This study utilized nano-LC-MS/MS to integrate quantitative proteomic and N-glycomic analyses of both cells and EVs derived from the MDA-MB-231 breast cancer cell line cultured under normoxic and hypoxic conditions. Whole N-glycome and proteome profiling revealed that hypoxia has an impact on the asparagine N-linked glycosylation patterns and on the glycolysis/gluconeogenesis proteins in cells in terms of altered N-glycosylation for their adaptation to low-oxygen conditions. Distinct N-glycan types, high-mannose glycans like Man3 and Man9, were highly abundant in the hypoxic cells. On the other hand, alterations in the sialylation and fucosylation patterns were observed in the hypoxic cells. Furthermore, hypoxia-induced EVs exhibit a signature consisting of mono-antennary structures and specific N-glycans (H4N3F1S2, H3N3F1S0, and H7N4F3S2; H8N4F1S0 and H8N6F1S2), which are significantly associated with poor prognoses for breast tumors, presumably altering the interactions within the tumor microenvironment to promote tumorigenesis and metastasis. Our findings provide an overview of the N-glycan profiles, particularly under hypoxic conditions, and offer insights into the potential biomarkers for tracking tumor microenvironment dynamics and for developing precision medicine approaches in oncology.
Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Polissacarídeos , Proteoma , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vesículas Extracelulares/metabolismo , Feminino , Linhagem Celular Tumoral , Proteoma/metabolismo , Polissacarídeos/metabolismo , Glicosilação , Microambiente Tumoral , Hipóxia Celular , Proteômica/métodos , Espectrometria de Massas em Tandem , Glicômica/métodosRESUMO
BACKGROUND: During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it. METHODS: Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TßR2 and TßR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis. RESULTS: Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-ß1/Smad2/3 signaling, especially increased interaction of TßR2 and TßR1. Ultimately, CD9 was determined to directly affect TßR1-TßR2 association in hypoxic fibroblast. CONCLUSION: Collectively, these findings suggest that CD9 promotes TßR2-TßR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.
Assuntos
Hipóxia Celular , Fibroblastos , Miofibroblastos , Tetraspanina 29 , Humanos , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 29/genética , Camundongos , Animais , Derme/citologia , Derme/metabolismo , Transdução de Sinais , Cicatrização , Pele/metabolismo , Pele/citologia , Hipóxia/metabolismo , Hipóxia/genéticaRESUMO
Hypoxia occurs in 90% of solid tumors and is associated with metastasis and mortality. Breast cancer cells that experience intratumoral hypoxia are 5x more likely to develop lung metastasis in animal models. Using spatial transcriptomics, we determine that hypoxic cells localized in more oxygenated tumor regions (termed 'post-hypoxic') retain expression of hypoxia-inducible and NF-kB-regulated genes, even in the oxygen-rich bloodstream. This cellular response is reproduced in vitro under chronic hypoxic conditions followed by reoxygenation. A subset of genes remains increased in reoxygenated cells. MUC1/MUC1-C is upregulated by both HIF-1α and NF-kB-p65 during chronic hypoxia. Abrogating MUC1 decreases the expression of superoxide dismutase enzymes, causing reactive oxygen species (ROS) production and cell death. A hypoxia-dependent genetic deletion of MUC1, or MUC1-C inhibition by GO-203, increases ROS levels in circulating tumor cells (CTCs), reducing the extent of metastasis. High MUC1 expression in tumor biopsies is associated with recurrence, and MUC1+ CTCs have lower ROS levels than MUC1- CTCs in patient-derived xenograft models. This study demonstrates that therapeutically targeting MUC1-C reduces hypoxia-driven metastasis.
Assuntos
Neoplasias da Mama , Mucina-1 , Espécies Reativas de Oxigênio , Mucina-1/metabolismo , Mucina-1/genética , Humanos , Animais , Espécies Reativas de Oxigênio/metabolismo , Feminino , Linhagem Celular Tumoral , Camundongos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo , Metástase Neoplásica , Hipóxia/metabolismo , Hipóxia CelularRESUMO
The oxygen level in the tumor microenvironment (TME) plays a critical role in regulating cell fates such as proliferation, migration, apoptosis, and so forth. To better elucidate how hypoxia affects tumor cell behaviors, a series of microfluidic strategies have been utilized to generate an oxygen gradient covering both hypoxia and normoxia conditions. However, in most studies, some chemicals are introduced into microfluidic chips, causing the potential of their poor biocompatibility. The common oxygen gradient with linear variation does not allow the effects of specific oxygen concentrations on tumor cells to be analyzed accurately. In this paper, based on the physical method of gas diffusion, a microfluidic device integrated with an oxygen gradient generator is proposed for investigating effects of different hypoxia levels on responses of tumor cells. This device consists of three layers, i.e., upper layer, thin film layer, and bottom layer. The upper layer is used for introducing the initial gas and generating an oxygen gradient in the form of gas. The bottom layer is used for introducing cells and culture medium. The thin film layer separates the former two layers, allowing the gas to diffuse from the top to the bottom through it. The oxygen gradient in the bottom layer is finally generated in the form of dissolved oxygen. The device is fabricated using microfabrication technology. The effects of structural and working parameters of the device on the oxygen gradient are evaluated by finite element simulation. The oxygen gradient in cell culture channels is characterized by using oxygen-sensitive fluorescence materials. The proliferation and morphology of HeLa cells under specific oxygen levels are compared after culturing for 48 h. The oxygen gradient with a ladder-like distribution demonstrates that this microfluidic device can provide a prospective experimental platform for in vitro cell studies and revelation of the mechanism of tumor metastasis associated with a specific hypoxic microenvironment.
Assuntos
Oxigênio , Humanos , Oxigênio/química , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Células HeLa , Microambiente Tumoral , Hipóxia CelularRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a low 5-year survival rate of only 13%. Despite intense research efforts, PDAC remains insufficiently understood. In part, this is attributed to opposing effects of key players being unraveled, including the stroma but also molecules that act in a context-dependent manner. One such molecule is the transcription factor C/EBPδ, where we recently showed that C/EBPδ exerts tumor-suppressive effects in PDAC cells in vitro. To better understand the role of C/EBPδ in different contexts and the development of PDAC, we here build on these findings and assess the effect of C/EBPδ in a PDAC model in mice. We establish that the lack of oxygen in vivo-hypoxia-counteracts the tumor-suppressive effects of C/EBPδ, and identify a reciprocal feedback loop between C/EBPδ and HIF-1α. RNA sequencing of C/EBPδ-induced cells under hypoxia also suggests that the growth-limiting effects of C/EBPδ decrease with oxygen tension. Consequently, in vitro proliferation assays reveal that the tumor-suppressive activities of C/EBPδ are abrogated due to hypoxia. This study demonstrates the importance of considering major physiological parameters in preclinical approaches.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT , Carcinoma Ductal Pancreático , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Pancreáticas , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Camundongos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Humanos , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proliferação de Células , Hipóxia/metabolismo , Hipóxia Celular , Regulação Neoplásica da Expressão GênicaRESUMO
Acute myocardial infarction (MI) is a sudden, severe cardiac ischemic event that results in the death of up to one billion cardiomyocytes (CMs) and subsequent decrease in cardiac function. Engineered cardiac tissues (ECTs) are a promising approach to deliver the necessary mass of CMs to remuscularize the heart. However, the hypoxic environment of the heart post-MI presents a critical challenge for CM engraftment. Here, we present a high-throughput, systematic study targeting several physiological features of human induced pluripotent stem cell-derived CMs (hiPSC-CMs), including metabolism, Wnt signaling, substrate, heat shock, apoptosis, and mitochondrial stabilization, to assess their efficacy in promoting ischemia resistance in hiPSC-CMs. The results of 2D experiments identify hypoxia preconditioning (HPC) and metabolic conditioning as having a significant influence on hiPSC-CM function in normoxia and hypoxia. Within 3D engineered cardiac tissues (ECTs), metabolic conditioning with maturation media (MM), featuring high fatty acid and calcium concentration, results in a 1.5-fold increase in active stress generation as compared to RPMI/B27 control ECTs in normoxic conditions. Yet, this functional improvement is lost after hypoxia treatment. Interestingly, HPC can partially rescue the function of MM-treated ECTs after hypoxia. Our systematic and iterative approach provides a strong foundation for assessing and leveraging in vitro culture conditions to enhance the hypoxia resistance, and thus the successful clinical translation, of hiPSC-CMs in cardiac regenerative therapies.
Assuntos
Hipóxia Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Engenharia Tecidual/métodos , Medicina Regenerativa/métodos , Diferenciação Celular , Infarto do Miocárdio/terapia , Infarto do Miocárdio/metabolismo , Células CultivadasRESUMO
Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.
Assuntos
Células Epiteliais , Malato Desidrogenase , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos dos fármacos , Malato Desidrogenase/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/citologia , Hipóxia Celular/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Dano ao DNA , Apoptose/efeitos dos fármacosRESUMO
Background: Hypoxic pulmonary hypertension (HPH) is one of the important pathophysiological changes in chronic pulmonary heart disease. Hypoxia promotes the phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs). Extracellular exosomes regulate vascular smooth muscle cell (VSMC) phenotypic switch. Aim: Given the importance of exosomes and alveolar epithelial cells (AECs) in HPH, the present study aimed to address the issue of whether AEC-derived exosomes promote HPH by triggering PASMC phenotypic switch. Methods: Cell Counting Kit-8 (CCK-8), TRITC-phalloidin staining, and Western blotting were used to examine the effects of AEC-derived exosomes on cell proliferation, intracellular actin backbone distribution, and expression of phenotypic marker proteins in PASMCs. Transcriptomics sequencing was used to analyze differentially expressed genes (DEGs) between groups. Results: Hypoxia-induced exosomes (H-exos) could promote the proliferation of PASMCs, cause the reduction of cellular actin microfilaments, promote the expression of synthetic marker proteins (ELN and OPN), reduce the expression of contractile phenotypic marker proteins (SM22-α and α-SMA), and induce the phenotypic transformation of PASMCs. Transcriptomics sequencing analysis showed that the Rap1 signaling pathway was involved in the phenotypic transformation of PASMCs induced by H-exos. Conclusion: The present study identified that hypoxia-induced AEC-derived exosomes promote the phenotypic transformation of PASMCs and its mechanism is related to the Rap1 signaling pathway.
Assuntos
Proliferação de Células , Exossomos , Miócitos de Músculo Liso , Fenótipo , Artéria Pulmonar , Transdução de Sinais , Exossomos/metabolismo , Artéria Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Células Epiteliais Alveolares/metabolismo , Ratos , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Músculo Liso Vascular/metabolismo , Hipertensão Pulmonar/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Hipóxia/metabolismo , Hipóxia Celular/fisiologiaRESUMO
Lung cancer is the leading cause of cancer-related death globally. Metastasis is the most common reason of mortality in which hypoxia is suggested to have a pivotal role. However, the effect of hypoxia on the metastatic potential and migratory activity of cancer cells is largely unexplored and warrants detailed scientific investigations. Accordingly, we analyzed changes on cell proliferation and migratory activity both in single-cell migration and invasion under normoxic and hypoxic conditions in lung adenocarcinoma cell lines. Alterations in crucial genes and proteins associated with cellular response to hypoxia, epithelial-mesenchymal transition, proliferation and apoptosis were also analyzed. Generally, we observed no change in proliferation upon hypoxic conditions and no detectable induction of apoptosis. Interestingly, we observed that single-cell motility was generally reduced while invasion under confluent conditions using scratch assay was enhanced by hypoxia in most of the cell lines. Furthermore, we detected changes in the expression of EMT markers that are consistent with enhanced motility and metastasis-promoting effect of hypoxia. In summary, our study indicated cell line-, time of exposure- and migrational type-dependent effects of hypoxia in cellular proliferation, motility and gene expression. Our results contribute to better understanding and tackling cancer metastasis.
Assuntos
Adenocarcinoma de Pulmão , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Apoptose , Hipóxia Celular , Regulação Neoplásica da Expressão Gênica , Células A549RESUMO
BACKGROUND: Rapid proliferation and outgrowth of tumor cells frequently result in localized hypoxia, which has been implicated in the progression of lung cancer. The present study aimed to identify key long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in hypoxia-induced A549 lung cancer cells, and to investigate their potential underlying mechanisms of action. METHODS: High-throughput sequencing was utilized to obtain the expression profiles of lncRNA and mRNA in both hypoxia-induced and normoxia A549 lung cancer cells. Subsequently, a bioinformatics analysis was conducted on the differentially expressed molecules, encompassing functional enrichment analysis, protein-protein interaction (PPI) network analysis, and competitive endogenous RNA (ceRNA) analysis. Finally, the alterations in the expression of key lncRNAs and mRNAs were validated using real-time quantitative PCR (qPCR). RESULTS: In the study, 1155 mRNAs and 215 lncRNAs were identified as differentially expressed between the hypoxia group and the normoxia group. Functional enrichment analysis revealed that the differentially expressed mRNAs were significantly enriched in various pathways, including the p53 signaling pathway, DNA replication, and the cell cycle. Additionally, key lncRNA-miRNA-mRNA relationships, such as RP11-58O9.2-hsa-miR-6749-3p-XRCC2 and SNAP25-AS1-hsa-miR-6749-3p-TENM4, were identified. Notably, the qPCR assay demonstrated that the expression of SNAP25-AS1, RP11-58O9.2, TENM4, and XRCC2 was downregulated in the hypoxia group compared to the normoxia group. Conversely, the expression of LINC01164, VLDLR-AS1, RP11-14I17.2, and CDKN1A was upregulated. CONCLUSION: Our findings suggest a potential involvement of SNAP25-AS1, RP11-58O9.2, TENM4, XRCC2, LINC01164, VLDLR-AS1, RP11-14I17.2, and CDKN1A in the development of hypoxia-induced lung cancer. These key lncRNAs and mRNAs exert their functions through diverse mechanisms, including the competitive endogenous RNA (ceRNA) pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares , RNA Longo não Codificante , RNA Mensageiro , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células A549 , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Redes Reguladoras de Genes , Perfilação da Expressão Gênica , Mapas de Interação de Proteínas/genética , Biologia Computacional/métodos , Hipóxia Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Macrophages orchestrate tissue homeostasis and immunity. In the tumor microenvironment (TME), macrophage presence is largely associated with poor prognosis because of their reprogramming into immunosuppressive cells. We investigated the effects of hypoxia, a TME-associated feature, on the functional, epigenetic, and transcriptional reprogramming of macrophages and found that hypoxia boosts their immunogenicity. Hypoxic inflammatory macrophages are characterized by a cluster of proinflammatory genes undergoing ten-eleven translocation-mediated DNA demethylation and overexpression. These genes are regulated by NF-κB, while HIF1α dominates the transcriptional reprogramming, demonstrated through ChIP-seq and pharmacological inhibition. In bladder and ovarian carcinomas, hypoxic inflammatory macrophages are enriched in immune-infiltrated tumors, correlating with better patient prognoses. Coculture assays and cell-cell communication analyses support that hypoxic-activated macrophages enhance T cell-mediated responses. The NF-κB-associated hypomethylation signature is displayed by a subset of hypoxic inflammatory macrophages, isolated from ovarian tumors. Our results challenge paradigms regarding the effects of hypoxia on macrophages and highlight actionable target cells to modulate anticancer immune responses.
Assuntos
Reprogramação Celular , Proteínas de Ligação a DNA , Dioxigenases , Macrófagos , NF-kappa B , Proteínas Proto-Oncogênicas , Microambiente Tumoral , Humanos , Hipóxia Celular , Linhagem Celular Tumoral , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/metabolismo , Macrófagos/imunologia , NF-kappa B/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Microambiente Tumoral/imunologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genéticaRESUMO
BACKGROUND: Pulmonary hypertension is a rare, progressive disorder that can lead to right ventricular hypertrophy, right heart failure, and even sudden death. N6-methyladenosine modification and the main methyltransferase that mediates it, methyltransferase-like (METTL) 3, exert important effects on many biological and pathophysiological processes. However, the role of METTL3 in pyroptosis remains unclear. METHODS AND RESULTS: Here, we characterized the role of METTL3 and the underlying cellular and molecular mechanisms of pyroptosis, which is involved in pulmonary hypertension. METTL3 was downregulated in a pulmonary hypertension mouse model and in hypoxia-exposed pulmonary artery smooth muscle cell. The small interfering RNA-induced silencing of METTL3 decreased the m6A methylation levels and promoted pulmonary artery smooth muscle cell pyroptosis, mimicking the effects of hypoxia. In contrast, overexpression of METTL3 suppressed hypoxia-induced pulmonary artery smooth muscle cell pyroptosis. Mechanistically, we identified the phosphate and tension homology deleted on chromosome 10 (PTEN) gene as a target of METTL3-mediated m6A modification, and methylated phosphate and tension homology deleted on chromosome 10 mRNA was subsequently recognized by the m6A "reader" protein insulin-like growth factor 2 mRNA-binding protein 2, which directly bound to the m6A site on phosphate and tension homology deleted on chromosome 10 mRNA and enhanced its stability. CONCLUSIONS: These results identify a new signaling pathway, the METTL3/phosphate and tension homology deleted on chromosome 10/insulin-like growth factor 2 mRNA-binding protein 2 axis, that participates in the regulation of hypoxia-induced pyroptosis.
Assuntos
Adenosina , Modelos Animais de Doenças , Metiltransferases , Músculo Liso Vascular , Miócitos de Músculo Liso , PTEN Fosfo-Hidrolase , Artéria Pulmonar , Piroptose , Animais , Metiltransferases/metabolismo , Metiltransferases/genética , Artéria Pulmonar/patologia , Artéria Pulmonar/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos , Metilação , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Masculino , Camundongos Endogâmicos C57BL , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/metabolismo , Hipóxia/genética , Células Cultivadas , Humanos , Transdução de Sinais , Hipóxia Celular , Metilação de RNARESUMO
Pulmonary hypertension (PH) is a serious pulmonary vascular disease characterized by vascular remodeling. Circular RNAs (CircRNAs) play important roles in pulmonary hypertension, but the mechanism of PH is not fully understood, particularly the roles of circRNAs located in the nucleus. Circ-calmodulin 4 (circ-calm4) is expressed in both the cytoplasm and the nucleus of pulmonary arterial smooth muscle cells (PASMCs). This study aimed to investigate the role of endonuclear circ-calm4 in PH and elucidate its underlying signaling pathway in ferroptosis. Immunoblotting, quantitative real-time polymerase chain reaction (PCR), malondialdehyde (MDA) assay, immunofluorescence, iron assay, dot blot, and chromatin immunoprecipitation (ChIP) were performed to investigate the role of endonuclear circ-calm4 in PASMC ferroptosis. Increased endonuclear circ-calm4 facilitated ferroptosis in PASMCs under hypoxic conditions. We further identified the cartilage oligomeric matrix protein (COMP) as a downstream effector of circ-calm4 that contributed to the occurrence of hypoxia-induced ferroptosis in PASMCs. Importantly, we confirmed that endonuclear circ-calm4 formed circR-loops with the promoter region of the COMP gene and negatively regulated its expression. Inhibition of COMP restored the phenotypes related to ferroptosis under hypoxia stimulation combined with antisense oligonucleotide (ASO)-circ-calm4 treatment. We conclude that the circ-calm4/COMP axis contributed to hypoxia-induced ferroptosis in PASMCs and that circ-calm4 formed circR-loops with the COMP promoter in the nucleus and negatively regulated its expression. The circ-calm4/COMP axis may be useful for the design of therapeutic strategies for protecting cellular functionality against ferroptosis and pulmonary hypertension.
Assuntos
Ferroptose , Miócitos de Músculo Liso , Artéria Pulmonar , RNA Circular , Animais , Masculino , Camundongos , Proteína de Matriz Oligomérica de Cartilagem/genética , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Hipóxia Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Ferroptose/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Tumor hypoxia is associated with a poorer prognosis in cancer patients and can diminish the efficacy of radiation therapy (RT). This study investigates the potential of metformin to enhance radiosensitivity in hypoxic cancer cells. METHODS: Preliminary experiments were conducted to validate the impact of hypoxia on radiation response. Reactive oxygen species (ROS) levels, cell migration, and cell death were assessed in hypoxic, radiated cells treated with metformin. Proteomic and ontological analyses were employed to identify molecular targets associated with the radiosensitizing effect of metformin. Proteomic and ontological findings were validated through patient samples and in vitro studies. RESULTS: Metformin amplified cell death, induced DNA fragmentation, decreased cell migration, and elevated ROS levels in hypoxic, radiated cells. Proteomic analyses revealed that GAPDH and TAGLN2 were identified as pivotal targets linked to the radiosensitizing effect of metformin. Oral cancer patients exhibited elevated levels of TAGLN2 and reduced levels of GAPDH. Metformin downregulated TAGLN2 and upregulated GAPDH in hypoxic, radiated cells. Additionally, metformin reduced levels of mutated p53. CONCLUSIONS: This study suggests that metformin can enhance radiosensitivity in hypoxic cells, operating through modulation of GAPDH and TAGLN2. Furthermore, metformin effectively reduces mutated p53 levels in radiated cells under hypoxic conditions.
Assuntos
Carcinoma de Células Escamosas , Metformina , Neoplasias Bucais , Radiossensibilizantes , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Neoplasias Bucais/radioterapia , Radiossensibilizantes/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteômica , Gliceraldeído-3-Fosfato Desidrogenases , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora) , Hipóxia Celular/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacosRESUMO
Accumulation studies confirmed that oxidative stress caused by ischemia after myocardial infarction (MI) is an important cause of ventricular remodeling. Exosome secretion through hypoxic pretreatment adipose-derived mesenchymal stem cells (ADSCs) ameliorates myocardial damaging post-MI. However, if ADSCs exosome can improve the microenvironment and ameliorate cardiac damage post-MI still unknown. Next-generation sequencing (NGS) was used to study abnormally expressed circRNAs in hypoxic pretreatment ADSC exosomes (HExos) and untreated ADSC exosomes (Exos). Bioinformatics and luciferase reporting were used to elucidate interaction correlation related to circRNA, mRNA, and miRNA. HL-1 cells were used to analyze the reactive oxygen species (ROS) and apoptosis under hypoxic conditions using immunofluorescence and flow cytometry. An MI mouse model was constructed and the therapeutic effect of Exos was determined using immunohistochemistry, immunofluorescence, and ELISA. The results showed that HExos had a more pronounced treatment effect than ADSC Exos on cardiac damage amelioration after MI. NGS showed that circ-Stt3b plays a role in HExo-mediated cardiac damage repair after MI. Overexpression of circ-Stt3b decreased apoptosis, ROS level, and inflammatory factor expression in HL-1 cells under hypoxic conditions. Bioinformatics and luciferase reporting data validated miR-15a-5p and GPX4 as downstream circ-Stt3b targets. GPX4 downregulation or miR-15a-5p overexpression reversed protective effect regarding circ-Stt3b upon HL-1 cells after exposure to a hypoxic microenvironment. Overexpression of circ-Stt3b increased the treatment effect of ASDSC Exos on cardiac damage amelioration after MI. Taken together, the study results demonstrated that Exos from hypoxic pretreatment ADSCs ameliorate cardiac damage post-MI through circ-Stt3b/miR-15a-5p/GPX4 signaling activation and decreased ferroptosis.