ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR-α-INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS

Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor-α (TNF-α) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF-α-induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF-α in a dose-dependent manner. UVB but not TNF-α induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF-α-induced apoptosis through the induction of TNFRs, Fas, and Fas-L in murine fibroblasts.     

细胞色素P450表氧化酶对肿瘤坏死因子诱导的内皮细胞凋亡的影响

目的研究细胞色素P450表氧化酶对肿瘤坏死因子诱导的内皮细胞(bovine aortic endothelial cells, BAECs)凋亡的影响.方法在原代培养的BAECs中,分别转染构建在腺相关病毒载体内的细胞色素P450表氧化酶基因2J2,2C11,F87V两周后,用肿瘤坏死因子(TNF-α)(10ng/ml)和放线菌素D(ActD)(5ng/ml)诱导内皮细胞凋亡,通过细胞形态学和流式细胞计数观察其凋亡变化,同时采用Western blot分析检测Bcl-2和P38和丝裂原激动蛋白激酶ERK的表达水平.结果与对照相比,转染细胞色素P450表氧化酶基因后BAECs 凋亡细胞数减少,Bcl-2表达增加, P38表达减低, ERK的磷酸化水平增加.结论细胞色素P450表氧化酶能明显的抑制肿瘤坏死因子诱导的BAECs凋亡,并可能通过上调Bcl-2、下调P38的表达水平和通过增加ERK的磷酸化水平发挥抗凋亡作用.     

MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20 ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca²⁺concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca²⁺-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca²⁺regulator or antioxidant, preventing lipid peroxidation in the process.     

Micro RNA‐550a interferes with vitamin D metabolism in peripheral B cells of patients with diabetes

The pathogenesis of diabetes is to be further investigated. Vitamin D3 (VitD3) can improve diabetes. Micro RNAs (miR) are involved in regulating cell activities. This study tests a hypothesis that miR‐550a interferes with the metabolism of VitD3 in peripheral B cells. In this study, blood samples were collected from patients with diabetes and healthy persons. The B cells were isolated from the blood samples to be treated with tumor necrosis factor (TNF)‐α. The B cells were then collected and analyzed for the expression of miR‐550a and cyp27b1. The results showed that B cells from healthy subjects were capable of converting VitD metabolite calcidiol to calcitriol, which was impaired in B cells collected from diabetic patients. The diabetic patients showed lower bone mineral density than that in healthy subject. The miR‐550a was negatively correlated with bone mineral density and the Levels of cyp27b1 in peripheral B cells of patients with diabetes. In vitro study showed that TNF‐α increased miR‐550a expression and inhibited the expression of cyp27b1 in B cells. miR‐550a mediated the effects of TNF‐α on inducing chromatin remodeling at the cyp27b1 gene locus. In conclusion, miR‐550a mediates the TNF‐α‐induced suppression of cyp27b1 expression in peripheral B cells of patients with diabetes, which can be blocked by inhibition of miR‐550a.     

Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway

Whether dendritic cell (DC) derived exosomes play a role in the progression of endothelial inflammation and atherosclerosis remains unclear. Using a transwell system and exosome release inhibitor GW4869, we demonstrated that mature DCs contributed to endothelial inflammation and exosomes were involved in the process. To further confirm this finding, we isolated exosomes from bone marrow dendritic cell (BMDC) culture medium (named DC‐exos) and stimulated human umbilical vein endothelial cell (HUVEC) with these DC‐exos. We observed that mature DC‐exos increased HUVEC inflammation through NF‐κB pathway in a manner similar to that of lipopolysaccharide. After a protein array analysis of exosomes, we identified and confirmed tumour necrosis factor (TNF)‐α on exosome membrane being the trigger of NF‐κB pathway in HUVECs. We then performed an in vivo study and found that the aorta endothelial of mice could uptake intravenously injected exosomes and was activated by these exosomes. After a period of 12 weeks of mature DC‐exos injection into ApoE?/? mice, the atherosclerotic lesions significantly increased. Our study demonstrates that mature DCs derived exosomes increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway. This finding extends our knowledge on how DCs affect inflammation and provides a potential method to prevent endothelial inflammation and atherosclerosis.     

Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK‐MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 μM; the half maximal cytotoxicity of doxorubicin was obtained at 3 μM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 μM and doxorubicin doses ≥1 μM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR‐1 blocking. Our results show doxorubicin‐stimulated production of TNFα, whereas enzastaurin‐stimulated TNFR‐1 expression on plasma membrane. The effect on TNFR‐1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin‐induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF‐α signal.     

Pulsatilla decoction and its active ingredients inhibit secretion of NO,ET‐1, TNF‐α, and IL‐1α in LPS‐induced rat intestinal microvascular endothelial cells

To investigate the pharmacological mechanism of the traditional Chinese medicine, Pulsatilla decoction (PD), the levels of nitric oxide (NO), endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNF‐α), and interleukin‐1α (IL‐1α) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with PD and its seven active ingredients, namely anemoside B₄, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with lipopolysaccharide (LPS) at 1 µg ml^?1 for 3 h and then treated with PD at 1, 5, and 10 mg ml^?1 and its seven ingredients at 1, 5, and 10 µg ml^?1 for 21 h, respectively. The results revealed that PD, anemonin, berberine, and esculetin inhibited the production of NO; PD, anemonin, and esculetin inhibited the secretion of ET‐1; PD, anemoside B₄, berberine, jatrorrhizine, and aesculin downregulated TNF‐α expression; PD, anemoside B₄, berberine, and palmatine decreased the content of IL‐1α. It showed that PD and its active ingredients could significantly inhibit the secretion of NO, ET‐1, TNF‐α, and IL‐1α in LPS‐induced RIMECs and suggested they would reduce inflammatory response via these cytokines. Copyright © 2009 John Wiley & Sons, Ltd.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

1950 MHz IMT‐2000 field does not activate microglial cells in vitro

Given the widespread use of the cellular phone today, investigation of potential biological effects of radiofrequency (RF) fields has become increasingly important. In particular, much research has been conducted on RF effects on brain function. To examine any biological effects on the central nervous system (CNS) induced by 1950 MHz modulation signals, which are controlled by the International Mobile Telecommunication‐2000 (IMT‐2000) cellular system, we investigated the effect of RF fields on microglial cells in the brain. We assessed functional changes in microglial cells by examining changes in immune reaction‐related molecule expression and cytokine production after exposure to a 1950 MHz Wideband Code Division Multiple Access (W‐CDMA) RF field, at specific absorption rates (SARs) of 0.2, 0.8, and 2.0 W/kg. Primary microglial cell cultures prepared from neonatal rats were subjected to an RF or sham field for 2 h. Assay samples obtained 24 and 72 h after exposure were processed in a blind manner. Results showed that the percentage of cells positive for major histocompatibility complex (MHC) class II, which is the most common marker for activated microglial cells, was similar between cells exposed to W‐CDMA radiation and sham‐exposed controls. No statistically significant differences were observed between any of the RF field exposure groups and the sham‐exposed controls in percentage of MHC class II positive cells. Further, no remarkable differences in the production of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and interleukin‐6 (IL‐6) were observed between the test groups exposed to W‐CDMA signal and the sham‐exposed negative controls. These findings suggest that exposure to RF fields up to 2 W/kg does not activate microglial cells in vitro. Bioelectromagnetics 31:104–112, 2010. © 2009 Wiley‐Liss, Inc.     

Mesenchymal stem cells modified to express lentivirus TNF‐α Tumstatin45–132 inhibit the growth of prostate cancer

Mesenchymal stem cells (MSCs) are a potential novel delivery system for cell‐based gene therapies. Although tumour necrosis factor (TNF)‐α has been shown to have antitumour activity, its use in therapy is limited by its systemic toxicity. For the present study, we designed lentivirus‐mediated signal peptide TNF‐α‐Tumstatin_45–132‐expressing mesenchymal stem cells (SPTT‐MSCs) as a novel anti‐cancer approach. We evaluated the effects of this approach on human prostate cancer cells (PC3 and LNCaP) by co‐culturing them with either SPTT‐MSCs or supernatants from their culture medium in vitro. The antitumour effects and possible mechanisms of action of SPTT‐MSCs were then determined in PC3 cells in vivo. The results showed that efficient TNF‐α‐Tumstatin_45–132‐expressing MSCs had been established, and demonstrated that SPTT‐MSCs inhibited the proliferation of and induced apoptosis in prostate cancer cells and xenograft tumours. As would be expected, given the properties of the individual proteins, the TNF‐α‐Tumstatin_45–132 fusion exerted potent cytotoxic effects on human prostate cancer cells and tumours via the death receptor‐dependent apoptotic pathway and via antiangiogenic effects. Our findings suggest that SPTT‐MSCs have significant activity against prostate cancer cells, and that they may represent a promising new therapy for prostate cancer.