Restored Cell Viability Research Articles

Homoharringtonine Exerts Anti-tumor Effects in Hepatocellular Carcinoma Through Activation of the Hippo Pathway.

Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer with a mortality rate of approximately 3–6/100,000 and is the third leading cause of cancer-related death worldwide. Although several small-molecule drugs have been developed for the treatment of HCC, the choice of an agent for patients who require systemic chemotherapy at an advanced stage is still limited. The Hippo pathway is an evolutionarily conserved tumor suppressive pathway commonly dysregulated in HCC, which makes it a promising target for anti-HCC therapies. Homoharringtonine (HHT) is an FDA-approved anti-leukemia drug with proven strong anti-tumor activity in solid tumors. In this study, we found that HHT could significantly inhibit HCC cell growth by suppressing cell proliferation and colony formation. Moreover, HHT repressed cell invasion and migration remarkably. Additionally, HHT induced cell cycle arrest at S phase and promoted apoptosis. Most importantly, we showed that HHT-induced apoptosis was a consequence of the Hippo pathway activation. Consistently, the MST1/2 inhibitor, XMU-MP-1, could restore cell viability and reverse HHT-induced cell apoptosis. Furthermore, in vivo results confirmed the tumor inhibitory effect of HHT. Taken together, our findings suggest that HHT is a potential alternative therapeutic agent for the treatment of HCC.

Feprazone Prevents Free Fatty Acid (FFA)-Induced Endothelial Inflammation by Mitigating the Activation of the TLR4/MyD88/NF-κB Pathway.

Increased levels of free fatty acid (FFA)-induced endothelial dysfunction play an important role in the initiation and development of atherosclerosis. Feprazone is a nonsteroidal anti-inflammatory compound. However, the beneficial effects of feprazone on FFA-induced endothelial dysfunction have not been reported before. In the current study, we found that treatment with feprazone ameliorated FFA-induced cell death of human aortic endothelial cells (HAECs) by restoring cell viability and reducing the release of lactate dehydrogenase (LDH). Importantly, we found that treatment with feprazone ameliorated FFA-induced oxidative stress by reducing the production of mitochondrial reactive oxygen species (ROS). In addition, feprazone prevented FFA-induced expression and secretion of proinflammatory cytokines and chemokines, such as chemokine ligand 5 (CCL5), interleukin-6 (IL-6), and interleukin-8 (IL-8). We also found that feprazone decreased the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Interestingly, we found that feprazone reduced the expression of cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). Our results also demonstrate that feprazone prevented FFA-induced activation of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa-B (NF-κB) signaling pathway. These findings suggest that feprazone might serve as a potential agent for the treatment of atherosclerosis by improving the endothelial function.

Melatonin Alleviates Hypoxia-Induced Apoptosis of Granulosa Cells by Reducing ROS and Activating MTNR1B-PKA-Caspase8/9 Pathway.

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT–MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

ABSTRACTv‐Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v‐Src‐mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities‐mediated chromosome instability. v‐Src directly phosphorylates Tyr‐15 of cyclin‐dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v‐Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v‐Src restores cancer cell viability reduced by various microtubule‐targeting agents (MTAs), although v‐Src does not alter cytotoxicity of DNA‐damaging anticancer drugs. v‐Src causes mitotic slippage of MTAs‐treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v‐Src also restores cell viability reduced by a polo‐like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v‐Src. These results suggest that the v‐Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src‐induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Mitochondrial Metabolism as Target of the Neuroprotective Role of Erythropoietin in Parkinson's Disease.

Existing therapies for Parkinson’s disease (PD) are only symptomatic. As erythropoietin (EPO) is emerging for its benefits in neurodegenerative diseases, here, we test the protective effect driven by EPO in in vitro (SH-SY5Y cells challenged by MPP+) and in vivo (C57BL/6J mice administered with MPTP) PD models. EPO restores cell viability in both protective and restorative layouts, enhancing the dopaminergic recovery. Specifically, EPO rescues the PD-induced damage to mitochondria, as shown by transmission electron microscopy, Mitotracker assay and PINK1 expression. Moreover, EPO promotes a rescue of mitochondrial respiration while markedly enhancing the glycolytic rate, as shown by the augmented extracellular acidification rate, contributing to elevated ATP levels in MPP+-challenged cells. In PD mice, EPO intrastriatal infusion markedly improves the outcome of behavioral tests. This is associated with the rescue of dopaminergic markers and decreased neuroinflammation. This study demonstrates cellular and functional recovery following EPO treatment, likely mediated by the 37 Kda isoform of the EPO-receptor. We report for the first time, that EPO-neuroprotection is exerted through restoring ATP levels by accelerating the glycolytic rate. In conclusion, the redox imbalance and neuroinflammation associated with PD may be successfully treated by EPO.

A Citrullus colocynthis fruit extract acutely enhances insulin-induced GLUT4 translocation and glucose uptake in adipocytes by increasing PKB phosphorylation

Ethnopharmacological relevanceCitrullus colocynthis (L.) Schrad is a common fruit in traditional medicine and used as remedy against various diseases, especially diabetes. Up to now, its anti-diabetic effects have been fully attributed to its enhancement of pancreatic insulin secretion. Whether C. colocynthis also ameliorates insulin action in peripheral tissues has not been investigated. Aim of the studyIn the present study, using 3T3-L1 adipocytes as cell model, we have investigated whether colocynth fruit extracts affect insulin action. Materials and methodsVarious extracts were prepared from the C. colocynthis fruit and screened using a cell-based 96 well plate GLUT4 translocation assay. Promising extracts were further studied for their effects on glucose uptake and cell viability. The effect on insulin signal transduction was determined by Western blot and the molecular composition was established by LC-MS. ResultsThe ethyl acetate fractions of aqueous non-defatted extracts of seed and pulp, designated Sna1 and Pna1, acutely enhanced insulin-induced GLUT4 translocation. In accordance, both extracts increased insulin-stimulated cellular glucose uptake. Pna1, which displayed greater effects on GLUT4 and glucose uptake than Sna1, was further investigated and was demonstrated to increase GLUT4 translocation without changing the half-maximum dose (ED50) of insulin, nor changing GLUT4 translocation kinetics. At the molecular level, Pna1 was found to enhance insulin-induced PKB phosphorylation without changing phosphorylation of the insulin receptor. Pna1 appeared not to be toxic to cells and, like insulin, restored cell viability during serum starvation. By investigating the molecular composition of Pna1, nine compounds were identified that made up 87% of the mass of the extract, one of which is likely to be responsible for the insulin-enhancing effects of Pna1. ConclusionsThe C. colocynthis fruit possesses insulin-enhancing activity. This activity may explain in part its anti-diabetic effects in traditional medicine. It also identifies the C. colocynthis as a source of a potential novel insulin enhancer that may prove to be useful to reduce hyperglycemia in type 2 diabetes.

Transcription factor Krüppel-like factor 5-regulated N-myc downstream-regulated gene 2 reduces IL-1β-induced chondrocyte inflammatory injury and extracellular matrix degradation

ABSTRACT Previous research has identified N-myc downstream-regulated gene 2 (NDRG2) as one of the differentially expressed genes common to rat models of osteoarthritis (OA) and human OA. The purpose of this study was to investigate the role of NDRG2 in OA. In this study, an in vitro OA model was constructed by challenging ATDC5 chondrocytes with 10 ng/ml IL-1β. After transfection of pcDNA3.1(+)/NDRG2, qPCR and western blot were performed to assay NDRG2 expression. The analyses of cell viability, apoptosis and inflammatory molecule expression were employed respectively by CCK-8, TUNEL and ELISA. The protein expression related to apoptosis, inflammation or extracellular matrix (ECM) degradation was detected by western blot. The binding of Krüppel-like factor 5 (KLF5) to NDRG2 promoter was verified by means of dual-luciferase reporter assay. After overexpression of both NDRG2 and KLF5 in IL-1β-stimulated ATDC5 chondrocytes, corresponding assays were performed to examine cell viability, apoptosis, inflammatory response and ECM degradation. In ATDC5 chondrocytes challenged with IL-1β, NDRG2 expression was much lower than that in the control group, whereas it’s overexpression helped restored cell viability and reduce cell apoptosis, inflammatory response and ECM degradation. It was also observed that KLF5 expression was decreased in IL-1β-stimulated ATDC5 chondrocytes, and that KLF5 bound to the NDRG2 promoter. Importantly, overexpressing KLF5 could reverse the protective effect of NDRG2 overexpression on IL-1β-stimulated ATDC5. Overall, NDRG2 could be transcriptionally regulated by transcription factor KLF5 and may play a protective role against chondrocyte the inflammatory response and ECM degradation in OA.

Reduction of anoxia-induced bioenergetic disturbance in astrocytes by methanol fruit extract of Tetrapleura tetraptera and in silico evaluation of the effect of its antioxidative constituents on excitotoxicity

Oxidative stress and excitotoxicity are some of the pathophysiological abnormalities in hypoxia-induced brain injury. This study evaluated the intrinsic antioxidant property of methanol fruit extract of Tetrapleura tetraptera (TT), traditionally used for managing brain diseases such as cerebral infarction in West Africa, and its ability to protect primary astrocytes from anoxia-induced cell death. The effect of the phytochemicals present in TT on excitotoxicity was assessed in silico, through docking with human glutamate synthetase (hGS). Chromatographic and spectrophotometric analyses of TT were performed. Primary astrocytes derived from neural stem cells were treated with TT and its effect on astrocyte viability was assessed. TT-treated astrocytes were then subjected to anoxic insult and, cell viability and mitochondrial membrane potential were evaluated. Molecular docking of hGS with detected phytochemicals in TT (aridanin, naringenin, ferulic acid, and scopoletin) was performed and the number of interactions with the lead compounds, aridanin, analyzed. HPLC-DAD analysis of TT revealed the presence of various bioactive phytochemicals. TT demonstrated notable antioxidant and radical scavenging activities. TT also protected astrocytes from anoxic insult by restoring cell viability and preventing alteration to mitochondrial membrane integrity. Aridanin, naringenin, ferulic acid, and scopoletin demonstrated good binding affinities with hGS indicating that Tetrapleura tetraptera is a potential source of new plant-based bioactives relevant in the therapy of neurodegenerative diseases.

Liposomes loaded with polyphenol-rich grape pomace extracts protect from neurodegeneration in a rotenone-basedin vitromodel of Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disease with no satisfactory therapy options. Similar to other neurodegenerative conditions, such as Alzheimer's and Huntington's diseases, oxidative stress plays a key factor in the neurodegeneration process. To counteract the uncontrolled increase of reactive oxygen species (ROS) and oxidative stress-dependent cell death, several preclinical and clinical tests exploit natural-derived organic antioxidants, such as polyphenols. Despite some promising results, free antioxidants show scarce brain accumulation and may exhaust their scavenging activity before reaching the brain. In this work, we developed an antioxidant therapeutic nanoplatform consisting of nano-sized functionalized liposomes loaded with selected polyphenol-rich vegetal extracts with high blood-brain barrier crossing capabilities. The antioxidant extracts were obtained from the grape seeds and skins as a byproduct of wine production (i.e., pomace), following a sustainable circular approach with reduced environmental impact. The antioxidant nanoplatform was successfully tested in a relevant in vitro model of PD, where it completely rescued the ROS levels, prevented the aggregation of α-synuclein fibrils, and restored cell viability, paving the way for preclinical translation of the approach.

The effects of oxygen and medicines on T cells in hypoxic co-culture

Objectives: Many patients with massive hemorrhage, respiratory failure due to trauma admit the emergency department, and further that the experience can fall into shock, inducing to sepsis, multiple organ failure due to hyperinflammation or immunosuppression. In the these patients, the low oxygen flow with immunosuppression is believed to play a significant role. Hence, oxygen supply and medicines is essential in severe trauma patients. Therefore, this study aims to investigate the effects of oxygen and variable medicines in hypoxic condition. Methods: T cells and macrophages were plated into trans-well plate for co-culture for 30 minutes in hypoxia. After that, the cells were stimulated with lipopolysaccharide (LPS) followed by variable medicines by normoxia or oxygen supply for 2 hrs and cells were inculated overnight under normoxic conditions. The T cell viability was measured by MTT, and the expression of interleukin-2 (IL-2), interleukin-8 (IL-8) and macrophage migration inhibitory factor (MIF) were measured by western blots using the T cells with co-culture with inflammatory maccrophages. Also, the concentration of MIF was analyzed by ELISA. Results: The T cells viability was decreased in hypoxia with LPS stimulation, however, pentoxifylline (PTX) effectively restored cell viability regardless of oxygen state (p < 0.05). Besides, PTX in oxygen supply status restored the decreases in IL-2 expression of T cells and the increases MIF in the LPS stimulation with hypoxia (p < 0.05). Conclusions: PTX has more effectively restored the T cells immunosuppression in hypoxia during oxygen supply, and has an immunomodulation effect by controlling hyperinflammation.

Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.

Activation of pannexin-1 mediates triglyceride-induced macrophage cell death

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin-1 activation, which increases extracellular ATP, leading to an increase in potassium efflux.

Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro.

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.

Urolithin A, a pomegranate metabolite, protects pancreatic β cells from apoptosis by activating autophagy

Ethnopharmacological relevanceUrolithin A is an active metabolite of plant polyphenol ellagic acid generated by intestinal flora, which is derived from strawberry or traditional anti-diabetic Chinese medicine such as Punica granatum L. and Phyllanthus emblica. The present study aimed to whether urolithin A can protect against glycolipid-toxicity-induced apoptosis of pancreatic β-cells and the underlying mechanisms. Materials and methodsApoptosis was induced in the pancreas of mice with type 2 diabetes and MIN6 pancreatic β-cells. CC-8 assay was conducted to determine cell viability. Flow cytometry, JC-1 fluorescent probe, and western blot assays were performed to assess apoptosis. Immunofluorescence and western blot assays were used to detect changes in autophagy. The mechanism of apoptosis was elucidated using autophagy inhibitor chloroquine. ResultsUrolithin A intervention significantly reduced pancreatic cell apoptosis in diabetic mice and MIN6 β cells. This was achieved by the downregulation of cleaved-caspase 3, cleaved-caspase 1, and restoration of cell viability, cell morphology and mitochondrial membrane potential, accompanied with the downregulation of autophagic protein SQSTM1/p62 and upregulation of LC3II. Chloroquine, an autophagy inhibitor, reversed the anti-glucolipotoxic and anti-apoptotic effects of urolithin A. ConclusionThese findings suggest that urolithin A protects against glucolipotoxicity-induced apoptosis in pancreatic β-cells by inducing activation of autophagy.

Trisulfide-Bearing PEG Brush Polymers Donate Hydrogen Sulfide and Ameliorate Cellular Oxidative Stress.

A potential approach to combat cellular dysfunction is to manipulate cell communication and signaling pathways to restore physiological functions while protecting unaffected cells. For instance, delivering the signaling molecule H2S to certain cells has been shown to restore cell viability and re-normalize cell behavior. We have previously demonstrated the ability to incorporate a trisulfide-based H2S-donating moiety into linear polymers with good in vitro releasing profiles and demonstrated their potential for ameliorating oxidative stress. Herein, we report two novel series of brush polymers decorated with higher numbers of H2S-releasing segments. These materials contain two trisulfide-based monomers co-polymerized with oligo(ethylene glycol methyl ether methacrylate) via reversible addition-fragmentation chain-transfer polymerization. The macromolecules were characterized to have a range of trisulfide densities with similar, well-defined molecular weight distribution, good H2S-releasing profiles, and high cellular tolerance. Using an amperometric technique, the H2S liberated and total sulfide release were found to depend on concentrations and chemical nature of triggering molecules (glutathione and cysteine) and, importantly, the position of reactive groups within the brush structure. Notably, when introduced to cells at well-tolerated doses, two macromolecular donors which have the same proportion as of the H2S-donating monomer (30%) but differ in releasing moiety location show similar cellular H2S-releasing kinetics. These donors can restore reactive oxygen species levels to baseline values, when polymer pretreated cells are exposed to exogenous oxidants (H2O2). Our work opens up a new aspect in preparing H2S macromolecule donors and their application to arresting cellular oxidative cascades.

Amitriptyline Protects Against Lidocaine-induced Neurotoxicity in SH-SY5Y Cells via Inhibition of BDNF-mediated Autophagy.

Amitriptyline (AMI) is a traditional tricyclic antidepressant that has been proven to exhibit neuroprotective effects in various neurological disorders. However, the underlying mechanism by which AMI attenuates lidocaine-induced neurotoxicity remains poorly understood. Brain-derived neurotrophic factor (BDNF) is an essential neurotrophin to neuronal development and survival in the brain, and recent studies have suggested that BDNF plays an important role in mediating lidocaine-induced neurotoxicity. The present study was performed to evaluate the protective effect of AMI against the neurotoxicity induced by lidocaine and to explore the role of BDNF-dependent autophagy in this process. The data showed that AMI pretreatment alleviated lidocaine-induced neurotoxicity, as evidenced by the restoration of cell viability, normalization of cell morphology, and reduction in the cell apoptosis index. In addition, autophagy inhibitor 3-methyladenine (3-MA) had a protective effect similar to that of AMI, but autophagy activator rapamycin eliminated the protective effect of AMI by suppressing mTOR activation. Moreover, at the molecular level, we found that AMI-mediated autophagy was involved in the expression of BDNF. The overexpression of BDNF or application of exogenous recombinant BDNF significantly suppressed autophagy and protected SH-SY5Y cells from apoptosis induced by Lido, whereas the neuroprotection of AMI was abolished by either knockdown of BDNF or use of a tropomyosin-related kinase B (TrkB) inhibitor ANA-12 in SH-SY5Y cells. Overall, our findings demonstrated that the protective effect of AMI against lidocaine-induced neurotoxicity correlated with inhibition of autophagy activity through upregulation of BDNF expression.

Apigenin causes necroptosis by inducing ROS accumulation, mitochondrial dysfunction, and ATP depletion in malignant mesothelioma cells.

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (ΔΨm), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)-histone H2A.X. and caused a transition delay at the G2/M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosis- and necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved ΔΨm loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.

Docosahexaenoic acid alleviates cell injury and improves barrier function by suppressing necroptosis signalling in TNF-α-challenged porcine intestinal epithelial cells.

Long-chain n-3 polyunsaturated fatty acids are known to have beneficial effects on intestinal health. However, the underling mechanisms are largely unknown. The present study was conducted to investigate whether docosahexaenoic acid (DHA) attenuates TNF-α-induced intestinal cell injury and barrier dysfunction by modulating necroptosis signalling. Intestinal porcine epithelial cell line 1 was cultured with or without 12.5 µg/ml DHA, followed by exposure to 50 ng/ml TNF-α for indicated time periods. DHA restored cell viability and cell number triggered by TNF-α. DHA also improved barrier function, which was indicated by increased trans-epithelial electrical resistance, decreased FD4 flux and increased membrane localisation of zonula occludins (ZO-1) and claudin-1. Moreover, DHA suppressed cell necrosis in TNF-α-challenged cells, as shown in the IncuCyte ZOOM™ live cell imaging system and transmission electron microscopy. In addition, DHA decreased protein expression of TNF receptor, receptor interacting protein kinase 1, RIP3 and phosphorylation of mixed lineage kinase-like protein, phosphoglycerate mutase family 5, dynamin-related protein 1 and high mobility group box-1 protein. Furthermore, DHA suppressed protein expression of caspase-3 and caspase-8. Collectively, these results indicate that DHA is capable of alleviating TNF-α-induced cell injury and barrier dysfunction by suppressing the necroptosis signalling pathway.

LncRNA SNHG7 sponges miR-449a to promote pituitary adenomas progression.

This study aimed to characterize the expression status and potentially mechanistic involvement of SNHG7 in pituitary adenoma. Relative expression of SNHG7 and miR-449a was analyzed by real-time PCR. Cell viability was measured with Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PI/Annexin V double staining followed by flow cytometry analysis. Cell invasion and migration were analyzed by wound healing and transwell assays, respectively. The regulatory action of miR-449a on SNHG7 was interrogated by luciferase reporter assay. We also investigated the pro-tumor activity of SNHG7 with the MMQ xenograft tumor mouse model. We identified the aberrant up-regulation of SNHG7 in pituitary adenoma both in vivo and in vitro, which associated with poor survival outcome. siRNA-mediated SNHG7-knockdown decreased cell viability, increased apoptosis and compromised migration and invasion. We further predicted and validated that SNHG7 negatively regulated miR-449a via sponging. Concurrent inhibition of miR-449a restored cell viability, apoptosis, migration and invasion influenced by SNHG7-deficiency. Most importantly, we demonstrated that SNHG7-silencing delayed xenograft tumor progression, which was accompanied with increased miR-449a and decreased Ki67 intensity. Our study highlighted the essential oncogenic properties of the SNHG7/miR-449a axis in pituitary adenoma.

High-glucose concentration aggravates TNF-alpha-induced cell viability reduction in human CD146-positive periodontal ligament cells via TNFR-1 gene demethylation.

Periodontitis is a chronic inflammatory disease that results in the destruction of periodontal soft tissue and the resorption of alveolar bone. Evidence indicates that in diabetic patients, hyperglycemia suppresses periodontal ligament stem cell (PDLSC) functions and leads to difficulties in periodontal repair. The present study aimed to explore the mechanisms by which high-glucose concentrations aggravate cell viability reduction in human CD146-positive PDLCs (CD146+ PDLCs) under tumor necrosis factor-alpha (TNF-alpha) induction. CD146+ PDLCs were isolated from periodontal ligament tissues and treated in the absence or presence of 10 ng/ml of TNF-alpha and 30 mM glucose. Cell viability was detected using Cell Counting Kit-8 assays and Luminescent Cell Viability Assays. Western blotting and real-time polymerase chain reaction were performed to determine tumor necrosis factor-alpha receptor-1 (TNFR-1) protein and messenger RNA expression. Bisulfite and MassArray methylation analyses were used to analyze the methylation status of the TNFR-1 gene. Our results indicated that cell viability was reduced after treatment with a combination of both high-glucose concentration and TNF-alpha. Treatment with 30 mM glucose suppressed DNA methyltransferase (DNMT) activities and DNMT1 protein expression, and this was accompanied by the upregulation of TNFR-1. Additionally, we found that the CpG island located within the TNFR-1 gene was hypomethylated under 30 mM glucose conditions. S-adenosylmethionine, an established methyl donor, reversed TNFR-1 upregulation and restored cell viability against high-glucose concentration and TNF-alpha. In conclusion, the present findings suggest that high-glucose-induced CpG island hypomethylation within the TNFR-1 gene plays an essential role in TNFR-1 upregulation, and this further enhances the cell viability reduction of CD146+ PDLCs caused by TNF-alpha.