1,25(OH)2 D3 inhibits Lewis lung cancer cell migration via NHE1-sensitive metabolic reprograming.

High prevalence and metastasis rates are characteristics of lung cancer. Glycolysis provides energy for the development and metastasis of cancer cells. The 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) has been linked to reducing cancer risk and regulates various physiological functions. We hypothesized that 1,25(OH)2 D3 could be associated with the expression and activity of Na+ /H+ exchanger isoform 1 (NHE1) of Lewis lung cancer cells, thus regulating glycolysis as well as migration by actin reorganization. Followed by online public data analysis, Vitamin D3 receptor, the receptor of 1,25(OH)2 D3 has been proved to be abundant in lung cancers. We demonstrated that 1,25(OH)2 D3 treatment suppressed transcript levels, protein levels, and activity of NHE1 in LLC cells. Furthermore, 1,25(OH)2 D3 treatment resets the metabolic balance between glycolysis and OXPHOS, mainly including reducing glycolytic enzymes expression and lactate production. In vivo experiments showed the inhibition effects on tumor growth as well. Therefore, we concluded that 1,25(OH)2 D3 could amend the NHE1 function, which leads to metabolic reprogramming and cytoskeleton reconstruction, finally inhibits the cell migration.

1,25(OH)2 D3 attenuates sleep disturbance in mouse models of Lewis lung cancer, in silico and in vivo.

Many clinical studies have reported that patients diagnosed with cancer will suffer from sleep disturbance during their clinical process, especially among lung cancer patients, and this effect will not easily subside. 1,25-dihydroxy-vitamin-D3 [1,25(OH)2 D3 ], the activated form of vitamin D, can participate in neuronal differentiation and prevent damage to the nervous system. However, little is known about the potential therapeutic effects of cancer-related psychiatric symptoms. In light of this, we hypothesized that a low circulating level of vitamin D was related to sleep quality in the presence of a tumor, 1,25(OH)2 D3 may be an effective way to ameliorate sleep disturbance and neurochemical alterations along with the cancer progress. Male C57BL/6 mice were implanted with intracranial transmitters to monitor electroencephalogram and were subcutaneously inoculated with Lewis lung cancer cells. The results demonstrated that on Days 19-20, tumor-bearing mice displayed fragmented sleep, shortened wake phase, prolonged sleep in the non-rapid eye movement phase, and the levels of vitamin D-associated genes in the brain had changed a lot compared to control mice. Importantly, 1,25(OH)2 D3 treatment really effectively saved the sleep quality of tumor-bearing mice. We further explored and confirmed that 1,25(OH)2 D3 repressed tumor-induced neuroinflammation (IL-1ß, TNF-α, IL-6, IL-10, IFN-γ, and IL-2), enhanced neurotrophic factors (brain-derived neurotrophic factor [BDNF], glialcellline-derived neurotrophic factor) and 5-HT system in the hippocampus, hypothalamus or cortex. A molecular docking approah manifested the ability of 1,25(OH)2 D3 to affect the activity of tryptophan hydroxylase 2 and BDNF. Together, our results suggested that 1,25(OH)2 D3 treatment may attenuate sleep disturbance in Lewis lung cancer-bearing mice, and become a promising strategy for treating cancer symptom clusters to ameliorate the quality of life of patients with cancer.

Rosthorin A inhibits non-small cell lung cancer cell growth and metastasis through repressing epithelial-mesenchymal transition via downregulating Slug.

Lung cancer always ranks first in the number of cancer deaths every year, accounting for 18.4% of total cancer deaths in 2018. Metastasis is the main cause of death in lung cancer patients. The identification of bioactive components of traditional Chinese medicine is very important for the development of novel reagents against non-small cell lung cancer (NSCLC). Rosthorin A has originated from Rabdosia rosthornii (Diels) Hara which excerpts from 'Chinese materia medica', and is known to have 'clear heat phlegm' properties in the folk. Little is known about the biological functions and mechanisms of Rosthorin A in cancer cells at present. The role of EMT in metastasis of a tumor cell is self-evident. Slug is an important EMT inducer, which is related to the development of lung cancer. Cell growth, clone assay, cell migration, cell invasion, and protein expression, and NSCLC transplanted tumor growth were performed in A549, H1299, and H1975 cells. Rosthorin A significantly inhibited the growth of NSCLC cells, it could prolong the survival of nude mice. Rosthorin A inhibited the migration and invasion of A549, H1299, and H1975 cells. Rosthorin A up-regulated E-cadherin expression level and down-regulated the expression of ß-catenin, N-cadherin, vimentin, Slug, and Twist. Rosthorin A could promote the expression of E-cadherin and inhibit the development of EMT by downregulating Slug, to inhibit the development and metastasis of NSCLC cells. In summary, Rosthorin A could be used as a promising candidate for the treatment of NSCLC patients with recurrence and metastasis.

Correlations between quantitative parameters of contrast-enhanced ultrasound and vasculogenic mimicry in murine tumor model: a novel noninvasive technique for assessment?

OBJECTIVE: Vasculogenic mimicry (VM) is a novel mechanism of tumor blood supply distinct from endothelial vessel (EV). VM is associated with malignancy, invasion, metastasis, and poor prognosis. Hitherto a noninvasive method for the assessment of VM in vivo has been lacking. METHODS: Contrast-enhanced ultrasound (CEUS) was performed to evaluate the quantitative parameters of tumors in mice. CD31 immunohistochemistry-Periodic Acid-Schiff double staining was conducted to identify the VM or EV in tumor tissues. Correlations between perfusion parameters and VM density was analyzed by Pearson correlation test. RESULTS: By the 15th day after tumor inoculation, the EV and VM density was 31.15 ± 7.14 and 14.11 ± 2.99 per 200× field. The maximal intensity (IMAX) was 301.19 ± 191.56%, and the rise time (RT), time to peak (TTP) and mean transit time (mTT) were 17.38 ± 7.82 s, 20.27 ± 9.61 s and 58.09 ± 26.44 s, respectively. VM density positively correlated to RT (r = 0.3598, P = 0.0226), TTP (r = 0.3733, P = 0.0177) and mTT(r = 0.6483, P <  0.0001), whereas EV density positively correlated to IMAX (r = 0.4519, P = 0.0034). The vascular diameter of VM was substantially larger than that of EV (43.81 ± 5.88 µm vs 11.21 ± 4.13 µm). CONCLUSION: Three quantitative parameters related to VM were obtained and the relationships between CEUS and VM were established. CEUS might thus provide a novel noninvasive method to assess VM in vivo.

Protective Effects and Mechanisms of Vaccarin on Vascular Endothelial Dysfunction in Diabetic Angiopathy.

Cardiovascular complications are a major leading cause of mortality in patients suffering from type 2 diabetes mellitus (T2DM). Vascular endothelial dysfunction is a core pathophysiological event in the early stage of T2DM and eventually leads to cardiovascular disease. Vaccarin (VAC), an active flavonoid glycoside extracted from vaccariae semen, exhibits extensive biological activities including vascular endothelial cell protection effects. However, little is known about whether VAC is involved in endothelial dysfunction regulation under high glucose (HG) or hyperglycemia conditions. Here, in an in vivo study, we found that VAC attenuated increased blood glucose, increased glucose and insulin tolerance, relieved the disorder of lipid metabolism and oxidative stress, and improved endothelium-dependent vasorelaxation in STZ/HFD-induced T2DM mice. Furthermore, in cultured human microvascular endothelial cell-1 (HMEC-1) cells, we showed that pretreatment with VAC dose-dependently increased nitric oxide (NO) generation and the phosphorylation of eNOS under HG conditions. Mechanistically, VAC-treated HMEC-1 cells exhibited higher AMPK phosphorylation, which was attenuated by HG stimulation. Moreover, HG-triggered miRNA-34a upregulation was inhibited by VAC pretreatment, which is in accordance with pretreatment with AMPK inhibitor compound C (CC). In addition, both reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) and VAC abolished HG-evoked dephosphorylation of AMPK and eNOS, increased miRNA-34a expression, and decreased NO production. These results suggest that VAC impedes HG-induced endothelial dysfunction via inhibition of the ROS/AMPK/miRNA-34a/eNOS signaling cascade.

Vaccarin administration ameliorates hypertension and cardiovascular remodeling in renovascular hypertensive rats.

Sympathetic overdrive, activation of renin angiotensin systems (RAS), and oxidative stress are vitally involved in the pathogenesis of hypertension and cardiovascular remodeling. We recently identified that vaccarin protected endothelial cell function from oxidative stress or high glucose. In this study, we aimed to investigate whether vaccarin attenuated hypertension and cardiovascular remodeling. Two-kidney one-clip (2K1C) model rats were used, and low dose of vaccarin (10 mg/kg), high dose of vaccarin (30 mg/kg), captopril (30 mg/kg) were intraperitoneally administrated. Herein, we showed that 2K1C rats exhibited higher systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), left ventricular mass/body weight ratio, myocardial hypertrophy or fibrosis, media thickness, and media thickness to lumen diameter, which were obviously alleviated by vaccarin and captopril. In addition, both vaccarin and captopril abrogated the increased plasma renin, angiotensin II (Ang II), norepinephrine (NE), and the basal sympathetic activity. The AT1R protein expressions, NADPH oxidase subunit NOX-2 protein levels and malondialdehyde (MDA) content were significantly increased, whereas superoxide dismutase (SOD) and catalase (CAT) activities were decreased in myocardium, aorta, and mesenteric artery of 2K1C rats, both vaccarin and captopril treatment counteracted these changes in renovascular hypertensive rats. Collectively, we concluded that vaccarin may be a novel complementary therapeutic medicine for the prevention and treatment of hypertension. The mechanisms for antihypertensive effects of vaccarin may be associated with inhibition of sympathetic activity, RAS, and oxidative stress.

Alkaline Cytosolic pH and High Sodium Hydrogen Exchanger 1 (NHE1) Activity in Th9 Cells.

CD4+ T helper 9 (Th9) cells are a newly discovered Th cell subset that produce the pleiotropic cytokine IL-9. Th9 cells can protect against tumors and provide resistance against helminth infections. Given their pivotal role in the adaptive immune system, understanding Th9 cell development and the regulation of IL-9 production could open novel immunotherapeutic opportunities. The Na+/H+ exchanger 1 (NHE1; gene name Slc9α1)) is critically important for regulating intracellular pH (pHi), cell volume, migration, and cell survival. The pHi influences cytokine secretion, activities of membrane-associated enzymes, ion transport, and other effector signaling molecules such as ATP and Ca2+ levels. However, whether NHE1 regulates Th9 cell development or IL-9 secretion has not yet been defined. The present study explored the role of NHE1 in Th9 cell development and function. Th cell subsets were characterized by flow cytometry and pHi was measured using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) dye. NHE1 functional activity was estimated from the rate of realkalinization following an ammonium pulse. Surprisingly, in Th9 cells pHi and NHE1 activity were significantly higher than in all other Th cell subsets (Th1/Th2/Th17 and induced regulatory T cells (iTregs)). NHE1 transcript levels and protein abundance were significantly higher in Th9 cells than in other Th cell subsets. Inhibition of NHE1 by siRNA-NHE1 or with cariporide in Th9 cells down-regulated IL-9 and ATP production. NHE1 activity, Th9 cell development, and IL-9 production were further blunted by pharmacological inhibition of protein kinase Akt1/Akt2. Our findings reveal that Akt1/Akt2 control of NHE1 could be an important physiological regulator of Th9 cell differentiation, IL-9 secretion, and ATP production.

DJ-1/Park7 Sensitive Na+ /H+ Exchanger 1 (NHE1) in CD4+ T Cells.

DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+ /H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pHi ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ-1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4+ T cells, and blunted the difference between DJ-1-/- and DJ-1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1-/- CD4+ T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc.

Interactions of TLR4 and PPARγ, Dependent on AMPK Signalling Pathway Contribute to Anti-Inflammatory Effects of Vaccariae Hypaphorine in Endothelial Cells.

Background /Aims: Accumulating evidence indicates that endothelial inflammation is one of the critical determinants in pathogenesis of atherosclerotic cardiovascular disease. Our previous studies had demonstrated that Vaccariae prevented high glucose or oxidative stress-triggered endothelial dysfunction in vitro. Very little is known about the potential effects of hypaphorine from Vaccariae seed on inflammatory response in endothelial cells. METHODS: In the present study, we evaluated the anti-inflammatory effects of Vaccariae hypaphorine (VH) on lipopolysaccharide (LPS)-challenged endothelial EA.hy926 cells. The inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1) were measured by real-time PCR (RT-PCR). The expressions of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), toll-like receptor 4 (TLR4), peroxisome proliferator-activated receptor γ (PPARγ) were detected by Western blotting or immunofluorescence. RESULTS: We showed that LPS stimulated the expressions of TNF-α, IL-1ß, MCP-1, VCAM-1 and TLR4, but attenuated the phosphorylation of AMPK and ACC as well as PPARγ protein levels, which were reversed by VH pretreatment. Moreover, we observed that LPS-upregulated TLR4 protein expressions were inhibited by PPARγ agonist pioglitazone, and the downregulated PPARγ expressions in response to LPS were partially restored by knockdown of TLR4. The negative regulation loop between TLR4 and PPARγ response to LPS was modulated by AMPK agonist AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) or A769662. CONCLUSIONS: Taken together, our results suggested that VH ameliorated LPS-induced inflammatory cytokines production in endothelial cells via inhibition of TLR4 and activation of PPARγ, dependent on AMPK signalling pathway.

1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells.

BACKGROUND/AIMS: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. METHODS: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. RESULTS: A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. CONCLUSIONS: In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)2D3 stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells.

BACKGROUND: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. METHODS: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. RESULTS: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. CONCLUSIONS: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

Salusin-ß mediates high glucose-induced endothelial injury via disruption of AMPK signaling pathway.

The dysregulated proliferation, migration, apoptosis and angiogenesis of endothelial cells are involved in diabetic endothelial dysfunction. The circulating salusin-ß levels were increased in diabetic patients, and salusin-ß contributes to diabetic cardiomyopathy in rats. However, the roles of salusin-ß in diabetes mellitus-induced endothelial dysfunction are not fully understood. Herein, we demonstrated the increased expressions of salusin-ß in human umbilical vein endothelial cells (HUVECs) cultured in HG medium. Exposure of HUVECs to HG inhibited the proliferation, migration, and angiogenesis, retarded cell cycle progression of endothelial cells, which were rescued by knockdown of salusin-ß. We also established that silencing of salusin-ß with adenoviruse-mediated shRNA reduced high glucose-induced apoptosis by up-regulating Bcl-2 expression and down-regulating Bax and caspase-3 expressions. Blockade of salusin-ß ameliorated HG-induced suppression of adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Of note, pretreatment with AMPK inhibitor Compound C abolished salusin-ß silencing-mediated endothelial protective effects. In summary, our results highlighted the involvement of salusin-ß in HG-related endothelial dysfunction, and salusin-ß contributed high glucose-induced endothelial injury via inactivation of AMPK signaling pathway.

C1q/TNF-Related Protein-9 Ameliorates Ox-LDL-Induced Endothelial Dysfunction via PGC-1α/AMPK-Mediated Antioxidant Enzyme Induction.

Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the critical determinants in endothelial dysfunction in many cardiovascular diseases such as atherosclerosis. C1q/TNF-related protein 9 (CTRP9) is identified to be an adipocytokine with cardioprotective properties. However, the potential roles of CTRP9 in endothelial function remain largely elusive. In the present study, the effects of CTRP9 on the proliferation, apoptosis, migration, angiogenesis, nitric oxide (NO) production and oxidative stress in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL were investigated. We observed that treatment with ox-LDL inhibited the proliferation, migration, angiogenesis and the generation of NO, while stimulated the apoptosis and reactive oxygen species (ROS) production in HUVECs. Incubation of HUVECs with CTRP9 rescued ox-LDL-induced endothelial injury. CTRP9 treatment reversed ox-LDL-evoked decreases in antioxidant enzymes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase quinone 1, and glutamate-cysteine ligase (GCL), as well as endothelial nitric oxide synthase (eNOS). Furthermore, CTRP9 induced activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α) and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Of interest, AMPK inhibition or PGC1-α silencing abolished CTRP9-mediated antioxidant enzymes levels, eNOS expressions, and endothelial protective effects. Collectively, we provided the first evidence that CTRP9 attenuated ox-LDL-induced endothelial injury by antioxidant enzyme inductions dependent on PGC-1α/AMPK activation.

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

BACKGROUND/AIMS: Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. METHODS: Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. RESULTS: ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-ß and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1ß, IL-6 and IL-17 but not IFN-γ mRNA levels. CONCLUSION: ASM is a negative regulator of natural and iTreg.

Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells.

BACKGROUND/AIMS: Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions. METHODS: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4. RESULTS: We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice. CONCLUSION: Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions.

Fruit Extract from Pyropolyporus fomentarius (L. ex Fr.) Teng Induces Mitochondria-Dependent Apoptosis in Leukemia Cells but Enhances Immunomodulatory Activities of Splenic Lymphocytes.

Pyropolyporus fomentarius (L. ex Fr.) Teng is a unique woody mushroom due to its medicinal value with numerous pharmacological activities. This study presented the potential antitumor and immunomodulatory properties of ethanol extract of P. fomentarius. The results showed that P. fomentarius extract inhibited the viability of murine leukemia L1210 cells in a dose-dependent manner with IC50 value of 69.35 µg/ml. Flow cytometry analysis demonstrated that the extract induced apoptosis in L1210 cells. Additionally, the decline of mitochondrial membrane potential was observed as well as the changes of caspase-3, caspase-9, Bcl-2, and Bax, suggesting that proapoptosis effect of the extract involved mitochondria-related pathway. Simultaneously, the P. fomentarius extract significantly enhanced the proliferation and activation of splenic lymphocytes in a dose-dependent manner. This P. fomentarius extract has potential applications as a natural antitumor agent with immunomodulatory activity.

Regulation of Na⁺/H⁺ Exchanger in Dendritic Cells by Akt1.

BACKGROUND/AIMS: Dendritic cells (DCs), antigen-presenting cells critically important for primary immune response and establishment of immunological memory, are activated by bacterial lipopolysaccharides (LPS) resulting in stimulation of Na(+)/H(+) exchanger, ROS formation and migration. The effects are dependent on phosphoinositide 3 (PI3) kinase and paralleled by Akt phosphorylation. The present study explored the contribution of the Akt isoform Akt1. METHODS: Cytosolic pH (pH(i)) (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence), Na(+)/H(+) exchanger activity (Na(+) dependent realkalinization after an ammonium pulse), cell volume (forward scatter in FACS analysis), and ROS production (2',7'-dichlorodihydrofluorescein diacetate [DCFDA] fluorescence) were determined in DCs isolated from bone marrow of mice lacking functional Akt1/PKBα (akt1(-/-)) and their wild type littermates (akt1(+/+)). RESULTS: Forward scatter was lower in akt1(-/-) than in akt1(+/+) DCs, whereas pH(i), Na(+)/H(+) exchanger activity and ROS formation were less in untreated akt1(-/-) and akt1(+/+) DCs. Exposure of DCs to LPS was followed by increase of forward scatter and ROS formation to a similar extent in akt1(-/-) and in akt1(+/+) DCs. A 4 hours treatment with either LPS (1µg/ml) or tert-butylhydroperoxide (tBOOH, 5 µM) significantly stimulated Na(+)/H(+) exchanger activity in both genotypes, effects, however, significantly blunted in akt1(-/-) DCs. CONCLUSION: The present observations demonstrate that Akt1 is required for the full stimulation of Na(+)/H(+) exchanger activity by LPS or oxidative stress in dendritic cells.

LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells.

OBJECTIVE: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H(+) and lactate. H(+) extrusion is in part accomplished by Na(+)/H(+) exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. METHODS: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pHi estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na(+)/H(+) exchanger activity from Na(+) dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. RESULTS: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na(+)/H(+) exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). CONCLUSIONS: LeftyA markedly down-regulates NHE1 expression, Na(+)/H(+) exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition.

Type-1 Na+/H+ exchanger is a prognostic factor and associate with immune infiltration in liver hepatocellular carcinoma.

AIMS: SLC9A1 plays an important role in the growth, differentiation and glycolysis of tumor cells. The present study aimed to elucidate the correlation between SLC9A1 and tumor immune infiltration. MAIN METHODS: Expression level of SLC9A1 gene in tumors was identified in GEPIA. The correlation between SLC9A1 and survival in various types of cancers was analyzed by the PrognoScan. SLC9A1 immune infiltration levels and clinical correlation analysis was generated via TIMER and TIMER2.0. KEGG enrichment analysis of SLC9A1 expression was evaluated via STRING. KEY FINDINGS: We found that, in cancers such as liver hepatocellular carcinoma (LIHC), the expression of SLC9A1 was significantly higher in tumor tissues compared with normal tissues, and was significantly associated with poor prognosis. Further analysis showed that SLC9A1 expression in LIHC was significantly positively correlated with immune cell infiltration, and the correlation was the highest for LIHC among 40 cancers. The expression of SLC9A1 is significantly correlated with the immune marker set of most immune cells in LIHC. Furthermore, we found that the expression level of TGF-ß (TGFB1) in Tregs showed the highest correlation with the expression of SLC9A1 in LIHC. SIGNIFICANCE: The increased expression of SLC9A1 is positively correlated with the prognosis of cancer and the level of immune infiltration. Therefore, SLC9A1 is an important prognostic factor for immunotherapy against hepatocellular carcinoma.

Vaccarin enhances intestinal barrier function in type 2 diabetic mice.

AIMS: Hyperglycemia and insulin resistance drive intestinal barrier dysfunction in type 2 diabetes (T2DM). Vaccarin, the main active component in the semen of traditional Chinese medicine Vaccaria has a definite effect on T2DM mice. The purpose of this study was to investigate whether vaccarin can enhance the intestinal barrier function in T2DM. MAIN METHODS: The T2DM mice model was established by streptozocin and high-fat diet. Vaccarin at a dose of 1 mg/kg/day was administered. We evaluated the effects of vaccarin on gut microbiota and intestinal barrier function by 16S rRNA sequencing, Western blot, quantitative fluorescent PCR (qPCR), and morphological observation. Moreover, we constructed a single layer of the human intestinal epithelium model to determine the effect of vaccarin in vitro. RESULTS: The experimental results showed that vaccarin alleviated inflammatory mediators in serum and intestinal tissue of mice (P < 0.05), which may depend on the improvement of tight junctions and gut microbiota (P < 0.05). Activation of extracellular regulated protein kinases (Erk1/2) stimulated myosin light chain kinase (MLCK). By inhibiting ERK expression (P < 0.05), vaccarin had similar effects to ERK inhibitors. In addition, the regulation of tight junction barriers also involved the abovementioned pathways in vivo. CONCLUSION: Vaccarin could protect the intestinal barrier by inhibiting the ERK/MLCK signaling pathway and modulate the composition of the microbiota. These results suggested that vaccarin may be an effective candidate for improving intestinal barrier changes in T2DM.