N6-methyladenosine (m6A) modification in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers of human, the tumor-related death of which ranks third among the common malignances. N6-methyladenosine (m6A) methylation, the most abundant internal modification of RNA in mammals, participates in the metabolism of mRNA and interrelates with ncRNAs. In this paper, we overviewed the complex function of m6A regulators in HCC, including regulating the tumorigenesis, progression, prognosis, stemness, metabolic reprogramming, autophagy, ferroptosis, drug resistance and tumor immune microenvironment (TIME). Furthermore, we elucidated the interplay between m6A modification and non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). Finally, we summarized the potential of m6A regulators as diagnostic biomarkers. What's more, we reviewed the inhibitors targeting m6A enzymes as promising therapeutic targets of HCC. We aimed to help understand the function of m6A methylation in HCC systematically and comprehensively so that more effective strategies for HCC treatment will be developed.

Metabolomics analysis reveals cytotoxic effects of ouabain towards psoriatic keratinocytes via impairment of glutathione metabolism.

Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to explore the effects of ouabain on proliferation and metabolism towards psoriatic keratinocytes. In human HaCaT keratinocytes, ouabain potently decreased viability, promoted apoptosis and caused G2/M cycle arrest. Metabolomics analysis indicated that ouabain markedly impaired glutathione metabolism. The solute carrier family 7 member 11 (SLC7A11) is an amino acid transporter highly specific to cysteine, which is critical for glutathione synthesis. Ouabain downregulated SLC7A11, reduced cysteine uptake and subsequently inhibited glutathione synthesis, probably through inhibiting Akt/mTOR/beclin axis that regulate protein activity of SLC7A11. The impaired glutathione synthesis and oxidative stress caused by ouabain may contribute to its cytotoxicity towards psoriatic keratinocytes. Our results provide experimental evidence supporting further study of ouabain as a potential anti-psoriatic agent.

Mesenchymal stromal cells promote the drug resistance of gastrointestinal stromal tumors by activating the PI3K-AKT pathway via TGF-ß2.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the prevailing sarcomas of the gastrointestinal tract. Tyrosine kinase inhibitors (TKIs) therapy, exemplified by Imatinib mesylate (IM), constitutes the established adjuvant therapy for GISTs. Nevertheless, post-treatment resistance poses a challenge that all patients must confront. The presence of tumor heterogeneity and secondary mutation mechanisms fail to account for some instances of acquired drug resistance. Certain investigations suggest a strong association between tumor drug resistance and mesenchymal stromal cells (MSC) in the tumor microenvironment, but the underlying mechanism remains obscure. Scarce research has explored the connection between GIST drug resistance and the tumor microenvironment, as well as the corresponding mechanism. METHODS: Immunofluorescence and fluorescence-activated cell sorting (FACS) methodologies were employed to detect the presence of MSC in GIST samples. The investigation encompassed the examination of MSC migration towards tumor tissue and the impact of MSC on the survival of GIST cells under IM treatment. Through ELISA, western blotting, and flow cytometry analyses, it was confirmed that Transforming Growth Factor Beta 2 (TGF-ß2) triggers the activation of the PI3K-AKT pathway by MSC, thereby facilitating drug resistance in GIST. RESULTS: Our findings revealed a positive correlation between a high proportion of MSC and both GIST resistance and a poor prognosis. In vitro studies demonstrated the ability of MSC to migrate towards GIST. Additionally, MSC were observed to secrete TGF-ß2, consequently activating the PI3K-AKT pathway and augmenting GIST resistance. CONCLUSIONS: Our investigation has revealed that MSC within GISTs possess the capacity to augment drug resistance, thereby highlighting their novel mechanism and offering a promising target for intervention in GIST therapy.

Metabolomic analysis of serum from pure coronary artery ectasia patients based on UPLC-QE/MS technique.

BACKGROUND: Coronary artery ectasia (CAE) is a cardiovascular disorder characterized by abnormal coronary artery dilation and disturbed coronary flow. The exact pathophysiology of CAE is still unclear. We aimed to investigate differences in metabolomic profiles between CAE patients and healthy controls. METHODS: Radial artery blood samples were collected from 14 pure CAE patients, 12 mixed CAE patients with atherosclerosis, and 14 controls with normal angiography. Differential serum metabolites were analyzed by untargeted ultra-high performance liquid chromatography-mass spectrometry. Serum ICAM-1, VEGF, ROS, and glutathione levels were also measured. RESULTS: Ten metabolites distinguished pure CAE patients from controls and mixed CAE, including 1-cyano-2-hydroxy-3-butene, 2,3-dihydro-6-methyl-5-(5-methyl-2-furanyl)-1H-pyrrolizine, 2-propionylpyrrole, 2-pyrrolidinone, 3-(2-furanylmethylene)pyrrolidine, D-alanine, furanofukinin, o-ethyltoluene, rotundine A, and SM(d18:1/18:1(9Z)). Related metabolic pathways include amino acid metabolism, sphingolipid dysfunction, energy metabolism, mitochondrial dysfunction, and oxidative stress. Serum concentrations of ICAM-1, VEGF and ROS were significantly elevated in CAE patients compared to controls, while glutathione decreased significantly in CAE patients. Moreover, ICAM-1 levels were negatively correlated with 2-propionylpyrrole, and VEGF levels were negatively correlated with SM(d18:1/18:1(9Z)), while GSH and ROS levels were correlated with the abundance of SM(d18:1/18:1(9Z)), further confirming systemic inflammation and oxidative stress in CAE. CONCLUSIONS: This is the first report describing differential serum metabolomic profiles of pure CAE patients compared to mixed CAE and healthy controls, which revealed 10 potential biomarkers that can provide an early diagnosis of pure CAE. These discriminatory metabolites and related metabolic pathways can help to better understand the pathogenesis of pure CAE.

ALKBH5-mediated N6-methyladenosine modification of TRERNA1 promotes DLBCL proliferation via p21 downregulation.

Long noncoding RNAs (lncRNAs) have crucial functions in the tumorigenesis and metastasis of cancers. N6-methyladenosine (m6A) modification of RNA is an important epigenetic regulatory mechanism in various malignancies. Nevertheless, the mechanism of m6A-modified lncRNA in diffuse large B cell lymphoma (DLBCL) has remained poorly defined. In the present study, we showed that lncRNA TRERNA1 was associated with the poor prognosis of DLBCL patients. TRERNA1 with internal m6A modification was highly correlated with the demethylase ALKBH5 expression. We further demonstrated that TRERNA1 was a potential downstream target of ALKBH5-mediated m6A modification by m6A-RNA sequencing and m6A-RIP assays. Decreased m6A methylation of TRERNA1 regulated by ALKBH5 was shown to regulate cell proliferation in vitro and in vivo. The results of mechanism analyses revealed that TRERNA1 recruited EZH2 to epigenetically silence the expression of the cyclin-dependent kinases inhibitor p21 by H3K27me3 modification of its promoter region. In addition, ALKBH5 further inhibited p21 expression. Taken together, our results elucidate the functional roles and epigenetic alterations of TRERNA1 through m6A modification in DLBCL. TRERNA1, the expression of which is upregulated by ALKBH5, acts as a scaffold that decreases p21 expression. The results of the present study provide novel targets for the diagnosis and treatment of DLBCL.

Dynamin-related protein 1 expression correlates with psoriasis disease severity and regulates keratinocyte function.

BACKGROUND: The pathogenesis of psoriasis is still not fully understood. Dynamin-related protein 1 (Drp1) regulates mitochondrial fission and is implicated in various inflammatory conditions, but research regarding Drp1 in the skin is scarce. OBJECTIVES: To examine Drp1 expression in psoriasis vulgaris lesional skin and the effect of Drp1 expression on keratinocyte proliferation and inflammatory mediator release in vitro. MATERIALS AND METHODS: Skin biopsies were collected, and the expression of Drp1 and TNF-α was investigated in the skin. Serum TNF-α level was also examined. The effect of Drp1 on keratinocyte proliferation and inflammatory mediator release was evaluated in HaCaT cells following Drp1 transfection. The effect of TNF-α on Drp1 expression was also studied in HaCaT cells. RESULTS: Drp1 expression was significantly increased and positively correlated with PASI score and TNF-α expression in skin. In HaCaT cells, Drp1 transfection altered cellular ATP and proliferation, induced G2/M arrest, and affected inflammatory mediator (TNF-α, IL-6 and CXCL8) release. Moreover, TNF-α induced Drp1 expression and recovered cellular ATP content and mediator release. CONCLUSION: Drp1 is significantly elevated in psoriasis vulgaris skin and positively correlates with disease severity, moreover, in vitro, Drp1 regulates keratinocyte function.

TNF stimulates IL-6, CXCL8 and VEGF secretion from human keratinocytes via activation of mTOR, inhibited by tetramethoxyluteolin.

Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and chronic inflammation. The pathogenesis of psoriasis involves proinflammatory cytokines, such as tumor necrosis factor (TNF), but the mechanism of keratinocyte activation is not well understood. Here, we show that TNF (10 or 50 ng/mL) stimulates a significant (P < .0001) gene expression and secretion of proinflammatory IL-6, CXCL8 and VEGF from both cultured human HaCaT and normal epidermal human keratinocytes (NHEKs). This effect occurs via activation of the mammalian target of rapamycin (mTOR) signalling complex as shown by Western blot analysis and phospho-ELISAs. Pretreatment with the novel natural flavonoid tetramethoxyluteolin (10-100 µmol L-1 ) significantly (P < .0001) inhibits gene expression and secretion (P < .0001) of all 3 mediators in a concentration-dependent manner. Moreover, tetramethoxyluteolin (50 µmol L-1 ) appears to be a potent inhibitor of the phosphorylated mTOR substrates (pmTORSer2448 , pp70S6KThr389 and p4EBP1Thr37/46 ) as compared to known mTOR inhibitors in keratinocytes. The present findings indicate that TNF stimulates skin inflammation via mTOR signalling. Inhibition by tetramethoxyluteolin may be used in the treatment for psoriasis.

Nanotube Formation: A Rapid Form of "Alarm Signaling"?

PURPOSE: Tunneling nanotubes (TNTs) are extremely thin (50-200 nm), actin-containing cell surface protrusions up to a few microns in length that can develop rapidly and connect various cell types. Mast cells (MCs) are unique immunomodulatory cells that are found perivascularly in all tissues. MCs communicate with many other cell types through the release of inflammatory, neurosensitizing, and vasoactive molecules, through which they are involved in the pathogenesis of many inflammatory diseases. We, therefore, investigated the possibility that MCs may form TNTs and communicate among themselves and with glioblastoma cells. METHODS: Laboratory Allergic Diseases (LAD)-2 human MCs were cultured in medium supplemented with 100 U/mL penicillin/streptomycin and 100 ng/mL recombinant human stem cell factor. They were incubated with 20 nmol/L deep red probe for 20 minutes and 50 nmol/L green probe for 30 minutes. Human glioblastoma cells were incubated with 20 nmol/L deep red probe only, moved to glass-bottom culture dishes, and observed using a substance P 2 confocal microscope. LAD2 MCs were stimulated with 2 µmol/L of the peptide substance P for 30 minutes at 37ºC. Confocal digital images were processed. FINDINGS: MCs can rapidly (within 5 minutes) form TNTs, which appear to transport mitochondrial and secretory granule particles among themselves and with cocultured glioblastoma cells. IMPLICATIONS: MCs are now accepted as having an important role in many diseases with an inflammatory component. TNTs provide a rapid and direct way for MCs to "alarm" other cell types with specificity not present when mediators are secreted into the tissue microenvironment. The identification of TNTs and their cargo could be important in the diagnosis and possible treatment of many inflammatory diseases.

The novel flavone tetramethoxyluteolin is a potent inhibitor of human mast cells.

BACKGROUND: Mast cells (MCs) are hematopoietic cells that mature in tissues and are involved in allergy, immunity, and inflammation by secreting multiple mediators. The natural flavone luteolin has anti-inflammatory actions and inhibits human mast cells (MCs). OBJECTIVE: We sought to investigate the ability of luteolin and its novel structural analog 3',4',5,7-tetramethoxyluteolin (methlut) to inhibit human MC mediator expression and release in vitro and in vivo. METHODS: Human LAD2 cells and umbilical primary human cord blood-derived cultured mast cells were stimulated with substance P (SP) or IgE/anti-IgE with or without preincubation with luteolin, methlut, or cromolyn (1-100 µmol/L) for 2 or 24 hours, after which mediator secretion was measured. The effect of the compounds on MC intracellular calcium levels and nuclear factor κB activation was also investigated. Pretreatment with methlut was also studied in mice passively sensitized with dinitrophenol-human serum albumin and challenged intradermally. RESULTS: Methlut is a more potent inhibitor than luteolin or cromolyn for ß-hexosaminidase and histamine secretion from LAD2 cells stimulated by either SP or IgE/anti-IgE, but only methlut and luteolin significantly inhibit preformed TNF secretion. Methlut is also a more potent inhibitor than luteolin of de novo-synthesized TNF from LAD2 cells and of CCL2 from human cord blood-derived cultured MCs. This mechanism of action for methlut might be due to its ability to inhibit intracellular calcium level increases, as well as nuclear factor κB induction, at both the transcriptional and translational levels in LAD2 cells stimulated by SP without affecting cell viability. Intraperitoneal treatment with methlut significantly decreases skin vascular permeability of Evans blue dye in mice passively sensitized to dinitrophenol-human serum albumin and challenged intradermally. CONCLUSION: Methlut is a promising MC inhibitor for the treatment of allergic and inflammatory conditions.

Luteolin inhibits human keratinocyte activation and decreases NF-κB induction that is increased in psoriatic skin.

Psoriasis (Ps) is an autoimmune disease characterized by keratinocyte hyperproliferation and chronic inflammation, with increased expression of tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Anti-TNF biologic agents are effective in treating Ps, but are associated with increased risk of infections and blood malignancies. Moreover, keratinocyte hyperproliferation and activation have yet to be addressed. Flavonoids, such as luteolin, are natural compounds with potent anti-inflammatory properties, but their actions on keratinocytes remain unknown. We show that TNF (50 ng/mL) triggers significant production of inflammatory mediators interleukin-6, interleukin-8 and VEGF from both human HaCaT and primary keratinocytes. Pretreatment with the flavonoid luteolin (10-100 µM) significantly inhibits mRNA expression and release of all three mediators in a concentration-dependent manner. More importantly, luteolin decreases TNF-induced phosphorylation, nuclear translocation and DNA binding of the nuclear factor-kappa B (NF-κB) typically involved in inflammatory mediator transcription. We also report that luteolin reduces TNF-induced mRNA expression of two genes (NFKB1 and RELA) encoding two NF-κB subunits (NF-κB p50 and NF-κB p65, respectively). Interestingly, we show that gene expression of RELA is increased in human psoriatic skin. Keratinocyte proliferation, which is a characteristic feature of psoriatic skin, is effectively reduced by luteolin in HaCaT cells, but not in primary keratinocytes. Finally, luteolin does not affect intracellular ATP production or viability. Appropriate formulations of luteolin and related flavones may be promising candidates to be developed into local and systemic treatments for Ps and other inflammatory skin diseases.

Mitochondria distinguish granule-stored from de novo synthesized tumor necrosis factor secretion in human mast cells.

BACKGROUND: Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve tumor necrosis factor (TNF). These cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete ß-hexosaminidase and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 h later. The regulation of these two distinct pathways is poorly understood. METHODS: Human LAD2 leukemic mast cells are stimulated by substance P. TNF secretion and gene expression were measured by ELISA and real-time PCR, and mitochondrial dynamics was observed in live cells under confocal microscopy. Cell energy consumption was measured in terms of oxygen consumption rate. RESULTS: Here, we show that granule-stored TNF is preformed, and its secretion from LAD2 mast cells stimulated by substance P (1) exhibits higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (2) shows rapid increase in intracellular calcium levels, and (3) exhibits reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide. This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells stimulated by an allergic trigger (IgE/streptavidin). CONCLUSION: Our findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes.

Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans.

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell "stabilizer", is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD(2). Que and cromolyn also inhibit histamine, leukotrienes and PGD(2) from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: relevance to atopic dermatitis.

BACKGROUND: Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE: We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS: Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS: Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION: Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.