ABSTRACT
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) have a variety of cardiovascular and renoprotective effects and have been developed as novel agents for the treatment of heart failure. However, the beneficial mechanisms of SGLT2i on cardiac tissue need to be investigated further. In this study, we established a mouse model of acute myocardial infarction (AMI) using coronary artery constriction surgery and investigated the role of dapagliflozin (DAPA) in protecting cardiomyocytes from hypoxic injury induced by AMI. In vitro experiments were done using hypoxic cultured H9c2 ventricular cells to verify this potential mechanism. Expression of the SIRT family and related genes and proteins was verified by qPCR, Western blotting and immunofluorescence staining, and the intrinsic potential mechanism of cardiomyocyte death due to AMI and hypoxia was comprehensively investigated by RNA sequencing. The RNA sequencing results of cardiomyocytes from AMI mice showed that the SIRT family may be mainly involved in the mechanisms of hypoxia-induced cardiomyocyte death. In vitro hypoxia-induced ventricular cells showed the role of dapagliflozin in conferring resistance to hypoxic injury in cardiomyocytes. It showed that SIRT1/3/6 were downregulated in H9c2 cells in a hypoxic environment, and the addition of dapagliflozin significantly increased the gene and protein expression of SIRT1, 3 and 6. We then verified the underlying mechanisms induced by dapagliflozin in hypoxic cardiomyocytes using RNA-seq, and found that dapagliflozin upregulated the hypoxia-induced gene downregulation, which includes ESRRA, EPAS1, AGTRAP, etc., that associated with SIRTs-related and apoptosis-related signaling to prevent H9c2 cell death. This study provides laboratory data for SGLT2i dapagliflozin treatment of AMI and confirms that dapagliflozin can be used to treat hypoxia-induced cellular necrosis in cardiomyocytes, in which SIRT1 and SIRT3 may play an important role. This opens up further opportunities for SGLT2i in the treatment of heart disease.
Subject(s)
Benzhydryl Compounds , Glucosides , Myocardial Infarction , Myocytes, Cardiac , Signal Transduction , Sirtuin 1 , Sodium-Glucose Transporter 2 Inhibitors , Glucosides/pharmacology , Glucosides/therapeutic use , Animals , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Benzhydryl Compounds/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Mice , Myocardial Infarction/metabolism , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , Sirtuin 1/metabolism , Sirtuin 1/genetics , Signal Transduction/drug effects , Male , Sirtuin 3/metabolism , Sirtuin 3/genetics , Sirtuins/metabolism , Sirtuins/genetics , Cell Line , Mice, Inbred C57BL , Disease Models, Animal , Cell Hypoxia/drug effects , Rats , Apoptosis/drug effectsABSTRACT
OBJECTIVES: Hypoxia is an important cause of chemotherapy resistance in gastric cancer. However, little is known about the growth of gastric cancer under purely hypoxia conditions. This study aims to study the effect of hypoxia on the growth patterns of gastric cancer cells and explore the response of gastric cancer cells to the chemotherapeutic drug 5-fluorouracil (5-FU) in a hypoxic environment. METHODS: Gastric cancer cells MKN45 were cultured under 1% oxygen hypoxia and conventional air conditions. An intervention group with the addition of the chemotherapeutic drug 5-FU was also established. The proliferation and apoptosis of gastric cancer cells under different oxygen conditions and intervention groups were detected using the cell counting kit-8 (CCK-8) method, JC-1 mitochondrial membrane potential assay, and Annexin-V/PI double staining method. Cell cycle changes were detected by flow cytometry, and mitochondrial changes were detected using electron microscopy. RESULTS: In the absence of 5-FU intervention, compared with the normoxia group, the hypoxia group showed higher rates of early and late apoptosis and higher cell death rates as indicated by the JC-1 mitochondrial membrane potential assay, Annexin-V/PI double staining, and CCK-8 results. Flow cytometry results showed that the cell cycle was arrested in the G0/G1 phase without progression. Electron microscopy revealed more severe mitochondrial destruction. However, with 5-FU intervention, the hypoxia group showed lower apoptosis rates, more cell cycle progression, and less mitochondrial destruction compared with the normoxia group. CONCLUSIONS: Hypoxic environments promote apoptosis and even death in gastric cancer cells, but hypoxia counteracts the efficacy of the chemotherapeutic drug 5-FU, which may contribute to 5-FU chemotherapy resistance.
Subject(s)
Apoptosis , Cell Hypoxia , Cell Proliferation , Fluorouracil , Membrane Potential, Mitochondrial , Stomach Neoplasms , Fluorouracil/pharmacology , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Humans , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Membrane Potential, Mitochondrial/drug effects , Cell Hypoxia/drug effects , Drug Resistance, Neoplasm , Cell Cycle/drug effects , Antimetabolites, Antineoplastic/pharmacologyABSTRACT
The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Carcinoma, Hepatocellular , Cell Proliferation , Hypoxia-Inducible Factor 1, alpha Subunit , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Cell Proliferation/drug effects , Cell Line, Tumor , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Isoquinolines/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Stress, Physiological/drug effects , Cell Hypoxia/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Mitochondria/metabolism , Mitochondria/drug effectsABSTRACT
BACKGROUND: Prostate cancer (PCa) ranks as the second most prevalent cancer in men, with advanced stages posing significant treatment challenges. Given its solid tumor nature, PCa is highly susceptible to hypoxia, a condition associated with resistance to radiation and chemotherapy, metastasis, and unfavorable patient outcomes. Hypoxia-inducible factors (HIFs) play a pivotal role in cancer cell adaptation to hypoxic environments, contributing to treatment resistance. Consequently, inhibitors targeting HIFs hold promise for cancer therapy. METHODS: In this study, we aimed to characterize novel HIF-1α inhibitors including Sodwanones A (1), B (2), C (3), G (4) and Yardenone 2 (5) isolated from marine sponges belonging to the Axinella genus. Our investigation evaluated the impact of these compounds on various aspects of HIF-1α regulation, including stabilization, nuclear localization, expression of HIF-1 target genes (while sparing HIF-2 target genes), cellular metabolism, as well as cell proliferation and viability in prostate cells under hypoxic conditions. RESULTS: Our findings revealed that among the compounds tested, Yardenone 2 exhibited notable effects in hypoxia: it destabilized HIF-1α at the protein level, decreased its nuclear localization, selectively altered the expression of HIF-1 target genes, and restrained cell proliferation in aggressive PC3 prostate cancer cells as well as in an MSK-PCa3 patient-derived organoid line. Moreover, it affected the morphology of these organoid. Yardenone 2 was also compared to Docetaxel, a specific microtubule inhibitor and a drug used in the treatment of prostate cancer. The comparison between the two compounds revealed notable differences, such as a lack of specificity to hypoxic cells of Docetaxel. CONCLUSION: These results mark the first demonstration that Yardenone 2 functions as a cytostatic-like inhibitor impacting microtubules, specifically targeting hypoxic cancer cells. This discovery suggests a promising avenue for novel therapeutic interventions in prostate cancer.
Subject(s)
Cell Proliferation , Hypoxia-Inducible Factor 1, alpha Subunit , Prostatic Neoplasms , Humans , Male , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Cell Proliferation/drug effects , Cell Line, Tumor , Animals , Porifera/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Antineoplastic Agents/pharmacology , Cell Hypoxia/drug effectsABSTRACT
BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
Subject(s)
Antioxidants , Extracellular Vesicles , Induced Pluripotent Stem Cells , Myocytes, Cardiac , NF-E2-Related Factor 2 , Proto-Oncogene Proteins c-akt , Signal Transduction , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Extracellular Vesicles/metabolism , NF-E2-Related Factor 2/metabolism , Humans , Proto-Oncogene Proteins c-akt/metabolism , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Signal Transduction/drug effects , Antioxidants/pharmacology , Oxidative Stress/drug effects , Cell Hypoxia/drug effects , Apoptosis/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , AnimalsABSTRACT
Photodynamic and photothermal therapies are promising treatments for cancer, dermatological, and ophthalmological conditions. However, photodynamic therapy (PDT) is less effective in oxygen-deficient tumor environments. Combining PDT with photothermal therapy (PTT) can enhance oxygen supply and treatment efficacy. Inorganic PTT agents pose toxicity risks, limiting their clinical use despite their high performance. In this study, we developed a novel nanomedicine integrating an all-organic photothermal agent and an organic photosensitizer, creating a colocalized nanoplatform to enhance phototherapy efficacy in cancer treatment. PTT nanoparticles (NPs) were synthesized through a thermal phase transition of organic chromophores, demonstrating superior photothermal properties and photostability. Utilizing this nanoplatform, we devised 'Combi NPs' for combined PDT-PTT nanomedicine. Tests on A549 cancer cell lines have revealed that Combi NPs exhibit superior cytotoxicity and induce apoptosis in hypoxic conditions, outperforming PTT-only NPs. The all-organic Combi NPs show significant potential for clinical cancer phototherapy in hypoxic microenvironments, potentially mitigating long-term nanomedicine accumulation and associated toxicity.
Subject(s)
Nanomedicine , Nanoparticles , Photochemotherapy , Photosensitizing Agents , Humans , Photochemotherapy/methods , Nanomedicine/methods , Photosensitizing Agents/pharmacology , Photosensitizing Agents/chemistry , Nanoparticles/chemistry , A549 Cells , Photothermal Therapy/methods , Apoptosis/drug effects , Combined Modality Therapy , Neoplasms/therapy , Neoplasms/drug therapy , Cell Line, Tumor , Cell Hypoxia/drug effects , Cell Survival/drug effectsABSTRACT
BACKGROUND: Pediatric postoperative cognitive dysfunction (POCD) is a prevalent complication following anesthesia and surgery. Hypoxia and propofol are the primary risk factors contributing to pediatric POCD. Our previous in vivo animal research has demonstrated that cognitive dysfunction in immature Sprague-Dawley (SD) rats, induced by hypoxia combined with propofol (HCWP), is closely associated with hippocampal neuron ferroptosis. METHODS AND RESULTS: In vivo transcriptome sequencing and KEGG functional analysis revealed significant enrichment of the mitophagy pathway. To further elucidate the relationship between mitophagy and ferroptosis, HT22 cells were selected to construct an in vitro HCWP model. Our findings indicate that HCWP activates excessive mitophagy in HT22 cells, leading to decreased mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) burst, mitochondrial fragmentation, and the induction of ferroptosis. To explore this causal relationship further, we employed Mdivi-1, a mitophagy inhibitor. Notably, low-dose Mdivi-1 (10 µM) effectively suppressed excessive mitophagy in HT22 cells, improved mitochondrial function and morphology, and mitigated markers associated with ferroptosis. The mechanism by which Mdivi-1 alleviates HCWP-induced ferroptosis in HT22 cells is likely due to its inhibition of excessive mitophagy, thereby promoting mitochondrial homeostasis. CONCLUSIONS: Our study suggests that mitophagy may be an upstream event in HCWP-induced ferroptosis in HT22 cells. Consequently, targeted regulation of mitophagy by Mdivi-1 may represent a promising approach to prevent cognitive dysfunction following HCWP exposure.
Subject(s)
Ferroptosis , Membrane Potential, Mitochondrial , Mitophagy , Propofol , Quinazolinones , Reactive Oxygen Species , Mitophagy/drug effects , Propofol/pharmacology , Ferroptosis/drug effects , Animals , Quinazolinones/pharmacology , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism , Rats , Mitochondria/drug effects , Mitochondria/metabolism , Cell Line , Hippocampus/metabolism , Hippocampus/drug effects , Hippocampus/pathology , Mice , Hypoxia/metabolism , Hypoxia/complications , Neurons/metabolism , Neurons/drug effects , Rats, Sprague-Dawley , Cell Hypoxia/drug effects , Postoperative Cognitive Complications/metabolismABSTRACT
AIMS: This study aimed to encapsulate natural killer (NK) cells in a hydrogel to sustain their function within the hypoxic tumour microenvironments. METHODS: An alginate-gelatine hydrogel was generated via electrospray technology. Hydrogel biocompatibility was assessed through cell counting kit-8 and Live/Dead assays to ascertain cell. Moreover, we analysed lactate dehydrogenase assays to evaluate the cytotoxicity against tumours and utilised RT-qPCR to analyse cytokine gene level. RESULTS: Alginate and gelatine formed hydrogels with diameters ranging from 489.2 ± 23.0 µm, and the encapsulation efficiency was 34.07 ± 1.76%. Encapsulated NK cells exhibited robust proliferation and tumour-killing capabilities under normoxia and hypoxia. Furthermore, encapsulation provided a protective shield against cell viability under hypoxia. Importantly, tumour-killing cytotoxicity through cytokines upregulation such as granzyme B and interferon-gamma was preserved under hypoxia. CONCLUSION: The encapsulation of NK cells not only safeguards their viability but also reinforces anticancer capacity, countering the inhibition of activation induced by hypoxia.
Subject(s)
Alginates , Cell Proliferation , Gelatin , Hydrogels , Killer Cells, Natural , Microspheres , Alginates/chemistry , Alginates/pharmacology , Hydrogels/chemistry , Hydrogels/pharmacology , Humans , Cell Proliferation/drug effects , Gelatin/chemistry , Cell Survival/drug effects , Cell Hypoxia/drug effects , Cell Encapsulation/methods , Animals , Tumor Microenvironment/drug effects , Cell Line, Tumor , MiceABSTRACT
Bone marrow-derived mesenchymal stem cells (MSCs), which are capable of differentiating into osteoblasts, are used in effective regenerative therapies. MSCs must be prompted to differentiate into osteoblasts for MSC transplantation to be effective. In this study, osteoblast differentiation markers involved in bone formation were evaluated to investigate the stress resistance of bone marrow-derived rat MSCs to dexamethasone and hypoxia and their ability to differentiate into osteoblasts. MSCs were allowed to differentiate into osteoblasts for 21 days in three different environments (dexamethasone treatment, hypoxic conditions [1% oxygen], or both). Osteoblast differentiation potential was evaluated according to alkaline phosphatase levels and a mineralisation assay. Immunofluorescence staining was used to determine the protein expression of the osteoblast differentiation markers type I collagen and osteopontin. MSCs differentiated into osteoblasts under hypoxic conditions but differentiated more slowly upon treatment with dexamethasone and dexamethasone plus hypoxia relative to the control. MSCs preconditioned with dexamethasone or hypoxia and then allowed to differentiate into osteoblasts under similar conditions differentiated comparably to control MSCs. MSCs that developed resistance to dexamethasone or hypoxia differentiated more quickly into osteoblasts than those that did not. The findings suggest that increasing the resistance of MSCs to stress by preconditioning them via dexamethasone or hypoxia exposure could result in more rapid differentiation into osteoblasts following transplantation.
Subject(s)
Cell Differentiation , Cell Hypoxia , Dexamethasone , Mesenchymal Stem Cells , Osteoblasts , Dexamethasone/pharmacology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Animals , Osteoblasts/drug effects , Osteoblasts/cytology , Osteoblasts/metabolism , Cell Differentiation/drug effects , Rats , Cell Hypoxia/drug effects , Osteogenesis/drug effects , Cells, Cultured , Alkaline Phosphatase/metabolism , Humans , Mesenchymal Stem Cell Transplantation/methods , Collagen Type I/metabolism , MaleABSTRACT
BACKGROUND: Tamoxifen (TAM) is a selective estrogen receptor modulator and has anti-estrogenic activity. Breast cancer cells acquire drug resistance to TAM as a consequence of long-term treatment. Lysophosphatidic acid (LPA) receptor-mediated signaling contributes to the promotion of tumor progression. This study aimed to evaluate the role of LPA receptors in the modulation of biological functions by long-term TAM treatment in breast cancer MCF-7 cells under hypoxic and estrogen-deprived conditions. METHODS: Long-term TAM treated (MCF-TAM) cells were generated from MCF-7 cells. Cells were cultured in estrogen-free medium at 1â¯% O2. LPA receptor expressions were measured by quantitative real-time RT-PCR analysis. Cell motile activity was investigated using Cell Culture Inserts. The CCK-8 kit was used to determine the cell proliferation rate. RESULTS: LPAR1 and LPAR3 expressions were elevated in MCF-TAM cells. MCF-TAM cell motility was enhanced by culturing at 1â¯% O2, compared with MCF-7 cells. When cells were cultured in estrogen-deprived medium at 1â¯% O2, the cell proliferation rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. CONCLUSION: These results suggest that LPA receptor-mediated signaling plays an important role in the acquisition of malignant properties in long-term TAM treated MCF-7 cells under hypoxic and estrogen-deprived conditions.
Subject(s)
Antineoplastic Agents, Hormonal , Breast Neoplasms , Cell Movement , Cell Proliferation , Receptors, Lysophosphatidic Acid , Signal Transduction , Tamoxifen , Humans , Receptors, Lysophosphatidic Acid/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/drug therapy , Tamoxifen/pharmacology , Signal Transduction/drug effects , MCF-7 Cells , Female , Cell Movement/drug effects , Cell Proliferation/drug effects , Antineoplastic Agents, Hormonal/pharmacology , Estrogens/metabolism , Estrogens/pharmacology , Cell Hypoxia/physiology , Cell Hypoxia/drug effectsABSTRACT
p53 has diversity functions in regulation of transcription, cell proliferation, cancer metastasis, etc. Recent studies have shown that p53 and nuclear factor-κB (NF-κB) co-regulate proinflammatory responses in macrophages. However, the role of p53 lysine lactylation (p53Kla) in mediating proinflammatory phenotypes in microglia under hypoxic conditions remains unclear. In the current study, we investigated the proinflammatory activation exacerbated by hypoxia and the levels of p53Kla in microglial cells. BV2 cells, an immortalized mouse microglia cell line, were divided into control, lipopolysaccharide (LPS)-induced, hypoxia (Hy), and LPS-Hy groups. The protein expression levels of p53 and p53Kla and the activation of microglia were compared among the four groups. Sodium oxamate and mutant p53 plasmids were transfected into BV2 cells to detect the effect of p53Kla on microglial proinflammatory activation. LPS-Hy stimulation significantly upregulated p53Kla levels in both the nucleus and the cytoplasm of BV2 cells. In contrast, the p53 protein levels were downregulated. LPS-Hy stimulation upregulated phosphorylated p65 protein levels in nuclear and activated the NF-κB pathway in BV2 cells, resulting in increased expression of pro-inflammatory cytokines (iNOS, IL6, IL1ß, TNFα), enhanced cell viability, and concomitantly, increased cytotoxicity. In conclusion, p53 lysine-lactylated modification contributes to LPS-induced proinflammatory activation in BV2 cells under hypoxia through NF-κB pathway and inhibition of lactate production may alleviate neuroinflammatory injury.
Subject(s)
Lipopolysaccharides , Lysine , Microglia , Tumor Suppressor Protein p53 , Animals , Lipopolysaccharides/pharmacology , Mice , Tumor Suppressor Protein p53/metabolism , Microglia/metabolism , Microglia/drug effects , Lysine/metabolism , Lysine/analogs & derivatives , Cell Line , Inflammation/metabolism , Inflammation/chemically induced , Inflammation/pathology , Cell Hypoxia/physiology , Cell Hypoxia/drug effects , NF-kappa B/metabolism , Cytokines/metabolismABSTRACT
Sixteen new dammarane-type triterpenoid saponins (1-16) featuring diverse structural variations in the side chain at C-17, along with twenty-one known analogues (17-37), have been isolated from the rhizomes of Gynostemma longipes C. Y. Wu, a plant renowned for its medicinal and edible properties. The structural elucidation of these compounds was accomplished through comprehensive analyses of 1D and 2D NMR and HRMS spectroscopic data, supplemented by comparison with previously reported data. Subsequent assays on the isolates for their protective effects against hypoxia-induced damage in pheochromocytoma cells (PC12 cells) revealed that nine saponins exhibited significant anti-hypoxic activities. Further investigation into the anti-hypoxia mechanisms of the representative saponins demonstrated that compounds 22 and 36 markedly reduced the levels of hypoxia-induced apoptosis. Additionally, these compounds were found to decrease the release of lactate dehydrogenase (LDH) and malondialdehyde (MDA), while increasing the activity of superoxide dismutase (SOD), thereby indicating that the saponins could mitigate hypoxia-induced injuries by ameliorating apoptosis and oxidative stress. These findings offer substantial evidence for the future utilization and development of G. longipes, identifying dammarane-type triterpenoid saponins as its active anti-hypoxic constituents.
Subject(s)
Apoptosis , Dammaranes , Gynostemma , Saponins , Triterpenes , PC12 Cells , Triterpenes/pharmacology , Triterpenes/chemistry , Gynostemma/chemistry , Rats , Animals , Apoptosis/drug effects , Molecular Structure , Saponins/pharmacology , Saponins/chemistry , Saponins/isolation & purification , Oxidative Stress/drug effects , Malondialdehyde/metabolism , Superoxide Dismutase/metabolism , Rhizome/chemistry , Cell Hypoxia/drug effects , Plant Extracts/pharmacology , Plant Extracts/chemistry , L-Lactate Dehydrogenase/metabolism , Protective Agents/pharmacology , Protective Agents/chemistryABSTRACT
Stroke and Alzheimer's disease (AD) are prevalent age-related diseases; however, the relationship between these two diseases remains unclear. In this study, we aimed to investigate the ability of melatonin, a hormone produced by the pineal gland, to alleviate the effects of ischemic stroke leading to AD by observing the pathogenesis of AD hallmarks. We utilized SH-SY5Y cells under the conditions of oxygen-glucose deprivation (OGD) and oxygen-glucose deprivation and reoxygenation (OGD/R) to establish ischemic stroke conditions. We detected that hypoxia-inducible factor-1α (HIF-1α), an indicator of ischemic stroke, was highly upregulated at both the protein and mRNA levels under OGD conditions. Melatonin significantly downregulated both HIF-1α mRNA and protein expression under OGD/R conditions. We detected the upregulation of ß-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression under both OGD and OGD/R conditions, while 10 µM of melatonin attenuated these effects and inhibited beta amyloid (Aß) production. Furthermore, we demonstrated that OGD/R conditions were able to activate the BACE1 promoter, while melatonin inhibited this effect. The present results indicate that melatonin has a significant impact on preventing the aberrant development of ischemic stroke, which can lead to the development of AD, providing new insight into the prevention of AD and potential stroke treatments.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Melatonin , Neuroblastoma , Melatonin/pharmacology , Humans , Alzheimer Disease/metabolism , Alzheimer Disease/etiology , Alzheimer Disease/pathology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Cell Line, Tumor , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Aspartic Acid Endopeptidases/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Glucose/metabolism , Amyloid beta-Peptides/metabolism , Oxygen/metabolism , Cell Hypoxia/drug effects , Hypoxia/metabolismABSTRACT
Introduction: Clinical studies have shown that endodontically-treated nonvital teeth exhibit less root resorption during orthodontic tooth movement. The purpose of this study was to explore whether hypoxic dental pulp stem cells (DPSCs) can promote osteoclastogenesis in orthodontically induced inflammatory root resorption (OIIRR). Methods: Succinate in the supernatant of DPSCs under normal and hypoxic conditions was measured by a succinic acid assay kit. The culture supernatant of hypoxia-treated DPSCs was used as conditioned medium (Hypo-CM). Bone marrow-derived macrophages (BMDMs) from succinate receptor 1 (SUCNR1)-knockout or wild-type mice were cultured with conditioned medium (CM), exogenous succinate or a specific inhibitor of SUCNR1 (4c). Tartrate-resistant acid phosphatase (TRAP) staining, Transwell assays, qPCR, Western blotting, and resorption assays were used to evaluate osteoclastogenesis-related changes. Results: The concentration of succinate reached a maximal concentration at 6 h in the supernatant of hypoxia-treated DPSCs. Hypo-CM-treated macrophages were polarized to M1 proinflammatory macrophages. Hypo-CM treatment significantly increased the formation and differentiation of osteoclasts and increased the expression of osteoclastogenesis-related genes, and this effect was inhibited by the specific succinate inhibitor 4c. Succinate promoted chemotaxis and polarization of M1-type macrophages with increased expression of osteoclast generation-related genes. SUCNR1 knockout decreased macrophage migration, M1 macrophage polarization, differentiation and maturation of osteoclasts, as shown by TRAP and NFATc1 expression and cementum resorption. Conclusions: Hypoxic DPSC-derived succinate may promote osteoclast differentiation and root resorption. The regulation of the succinate-SUCNR1 axis may contribute to the reduction in the OIIRR.
Subject(s)
Dental Pulp , Mice, Knockout , Osteoclasts , Osteogenesis , Root Resorption , Stem Cells , Succinic Acid , Animals , Mice , Dental Pulp/cytology , Dental Pulp/drug effects , Dental Pulp/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Root Resorption/pathology , Root Resorption/metabolism , Humans , Succinic Acid/metabolism , Osteogenesis/drug effects , Stem Cells/metabolism , Stem Cells/drug effects , Cell Differentiation/drug effects , Macrophages/metabolism , Macrophages/drug effects , Cell Hypoxia/drug effects , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Culture Media, Conditioned/pharmacology , Cells, CulturedABSTRACT
OBJECTIVE: This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. METHODS: The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 µM) or TRPV1 inhibitor capsazepine (1 µM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. RESULTS: After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. CONCLUSION: ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.
Subject(s)
Apoptosis , Calcium , Capsaicin/analogs & derivatives , Cell Survival , Ketamine , Myocytes, Cardiac , TRPV Cation Channels , TRPV Cation Channels/metabolism , TRPV Cation Channels/drug effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Calcium/metabolism , Cell Survival/drug effects , Apoptosis/drug effects , Animals , Ketamine/pharmacology , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/drug therapy , Rats , Capsaicin/pharmacology , Cell Hypoxia/drug effects , Cell Line , Flow Cytometry , Oxidative Stress/drug effects , Blotting, WesternABSTRACT
Inhibition of prolylhydroxylase-2 (PHD-2) in both normoxic and hypoxic cells is a critical component of solid tumours. The present study aimed to identify small molecules with PHD-2 activation potential. Virtually screening 4342 chemical compounds for structural similarity to R59949 and docking with PHD-2. To find the best drug candidate, hits were assessed for drug likeliness, antihypoxic and antineoplastic potential. The selected drug candidate's PHD-2 activation, cytotoxic and apoptotic potentials were assessed using 2-oxoglutarate, MTT, AO/EtBr and JC-1 staining. The drug candidate was also tested for its in-vivo chemopreventive efficacy against DMBA-induced mammary gland cancer alone and in combination with Tirapazamine (TPZ). Virtual screening and 2-oxoglutarate assay showed BBAP-6 as lead compound. BBAP-6 exhibited cytotoxic and apoptotic activity against ER+ MCF-7. In carmine staining and histology, BBAP-6 alone or in combination with TPZ restored normal surface morphology of the mammary gland after DMBA produced malignant alterations. Immunoblotting revealed that BBAP-6 reduced NF-κB expression, activated PHD-2 and induced intrinsic apoptotic pathway. Serum metabolomics conducted with 1H NMR confirmed that BBAP-6 prevented HIF-1α and NF-κB-induced metabolic changes in DMBA mammary gland cancer model. In a nutshell, it can be concluded that BBAP-6 activates PHD-2 and exhibits anticancer potential.
Subject(s)
Apoptosis , Breast Neoplasms , Hypoxia-Inducible Factor-Proline Dioxygenases , Humans , Female , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/prevention & control , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Apoptosis/drug effects , Mice , Cell Hypoxia/drug effects , Molecular Docking Simulation , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , MCF-7 Cells , Cell Line, Tumor , NF-kappa B/metabolism , Tirapazamine/pharmacology , Tirapazamine/chemistry , Tirapazamine/metabolismABSTRACT
Hypoxia-inducible factor (HIF)-1α is a crucial transcription factor associated with cancer metabolism and is regarded as a potent anticancer therapeutic strategy within the hypoxic microenvironment of cancer. In this study, stilbenoid derivatives were designed, synthesized, and assessed for their capacity to inhibit HIF-1α-associated cancer metabolism and evaluated for inhibition of cancer cell viability and HIF activation. Through the structure-activity relationship studies, compound 28e was identified as the most potent derivative. Specifically, under the hypoxic condition, 28e reduced the accumulation of HIF-1α protein and the expression of its target genes related to glucose metabolism without affecting the expression of HIF-1α mRNA. Furthermore, 28e inhibited glucose uptake, glycolytic metabolism, and mitochondrial respiration, decreasing cellular ATP production under hypoxic conditions. In addition, 28e displayed significant anti-tumor effects and effectively suppressed the accumulation of HIF-1α protein in tumor tissue in vivo xenograft model. These findings suggest that our stilbenoid derivatives exert their anticancer effects by targeting HIF-1α-centered cancer metabolism under hypoxic conditions.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit , Stilbenes , Animals , Humans , Mice , Antineoplastic Agents/pharmacology , Cell Hypoxia/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Glucose/metabolism , Glycolysis/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Mice, Inbred BALB C , Mice, Nude , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Stilbenes/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Glioblastoma multiforme (GBM) represents the deadliest tumor among brain cancers. It is a solid tumor characterized by uncontrolled cell proliferation generating the hypoxic niches in the cancer core. By inducing the transcription of hypoxic inducible factor (HIF), hypoxia triggers many signaling cascades responsible for cancer progression and aggressiveness, including enhanced expression of vascular endothelial growth factor (VEGF) or antioxidant enzymes, such as heme oxygenase-1 (HO-1). The present work aimed to investigate the link between HO-1 expression and the hypoxic microenvironment of GBM by culturing two human glioblastoma cell lines (U87MG and A172) in the presence of a hypoxic mimetic agent, deferoxamine (DFX). By targeting hypoxia-induced HO-1, we have tested the effect of a novel acetamide-based HO-1 inhibitor (VP18/58) on GBM progression. Results have demonstrated that hypoxic conditions induced upregulation and nuclear expression of HO-1 in a cell-dependent manner related to malignant phenotype. Moreover, our data demonstrated that the HO-1 inhibitor counteracted GBM progression by modulating the HIFα/HO-1/VEGF signaling cascade in cancer cells bearing more malignant phenotypes.
Subject(s)
Acetamides , Glioblastoma , Heme Oxygenase-1 , Signal Transduction , Vascular Endothelial Growth Factor A , Humans , Glioblastoma/metabolism , Glioblastoma/drug therapy , Glioblastoma/pathology , Heme Oxygenase-1/metabolism , Cell Line, Tumor , Acetamides/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Signal Transduction/drug effects , Brain Neoplasms/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Cell Proliferation/drug effects , Disease Progression , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Cell Hypoxia/drug effectsABSTRACT
Tumor cells in hypoxic conditions control cancer metabolism and angiogenesis by expressing HIF-1α. Tanshinone is a traditional Chinese medicine that has been shown to possess antitumor properties and exerts a therapeutic impact on angiogenesis. However, the precise molecular mechanism responsible for the antitumor activity of 3-Hydroxytanshinone (3-HT), a type of tanshinone, has not been fully understood. Therefore, our study aimed to investigate the mechanism by which 3-HT regulates the expression of HIF-1α. Our findings demonstrate that 3-HT inhibits HIF-1α activity and expression under hypoxic conditions. Additionally, 3-HT inhibits hypoxia-induced angiogenesis by suppressing the expression of VEGF. Moreover, 3-HT was found to directly bind to α-enolase, an enzyme associated with glycolysis, resulting in the suppression of its activity. This inhibition of α-enolase activity by 3-HT leads to the blockade of the glycolytic pathway and a decrease in glycolysis products, ultimately altering HIF1-α expression. Furthermore, 3-HT negatively regulates the expression of HIF-1α by altering the phosphorylation of AMP-activated protein kinase (AMPK). Our study's findings elucidate the mechanism by which 3-HT regulates HIF-1α through the inhibition of the glycolytic enzyme α-enolase and the phosphorylation of AMPK. These results suggest that 3-HT holds promise as a potential therapeutic agent for hypoxia-related angiogenesis and tumorigenesis.