Bioimaging analysis of nuclear factor-κB activity in Philadelphia chromosome-positive acute lymphoblastic leukemia cells reveals its synergistic upregulation by tumor necrosis factor-α-stimulated changes to the microenvironment

To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.     

An RNAi screen identifies USP2 as a factor required for TNF-α-induced NF-κB signaling

Tumor necrosis factor α (TNF-α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin-dependent processes are central to the regulation of TNF-α and nuclear factor κB (NF-κB) signaling. We performed a targeted siRNA screen for ubiquitin-specific proteases (USPs) and identified USP2 as a modulator of TNF-α-induced NF-κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes and IL-8 secretion. Our study also provides evidence for isoform-specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together, our results implicate USP2 as a novel positive regulator of TNF-α-induced NF-κB signaling and show that its expression is altered in tumor cells.     

Combination treatment using adenovirus vector-mediated tumor necrosis factor-alpha gene transfer and a NF-κB inhibitor for pancreatic cancer in mice

Gene therapy using an adenoviral vector expressing tumor necrosis factor-alpha (TNF-α) is a new therapeutic approach for pancreatic cancer. However, the efficacy of TNF-α is limited, because it activates nuclear factor-κB (NF-κB). We investigated the combined use of AxCAhTNF-α, adenoviral vector-expressing human TNF-α, and nafamostat mesilate, a serine-protease inhibitor, a NF-κB inhibitor, using pancreatic cancer in mice. In vitro, nafamostat mesilate inhibited TNF-α-induced NF-κB activation and enhanced TNF-α-induced apoptosis in human cancer cell line (MIAPaCa-2). In vivo, we assessed combination treatment of AxCAhTNF-α and nafamostat mesilate using xenograft models in nude mice by subcutaneous injection of MIAPaCa-2. When the implanted tumor size reached 8.0mm, TNF-α group (n=12), was injected AxCAhTNF-α intra-tumorally once a week, while combination group (n=12), was injected AxCAhTNF-α intra-tumorally once a week and nafamostat mesilate intraperitoneally thrice a week. In combination group, tumor growth was significantly slower, and the number of apoptosis cells was significantly greater than those in AxCAhTNF-α group (p<0.05). In conclusion, adenovirus vector-mediated TNF-α gene therapy combined with nafamostat mesilate was effective for pancreatic cancer in mice.     

Down-regulation of CYLD as a trigger for NF-κB activation and a mechanism of apoptotic resistance in hepatocellular carcinoma cells

The cylindromatosis gene (CYLD) was identified as a tumor suppressor gene, which is mutated in familial cylindromatosis (Brooke-Spiegler syndrome), an autosomal-dominant predisposition to multiple tumors of the skin appendages. CYLD is a deubiquitinating enzyme acting as a negative regulator of the nuclear factor κB (NF-κB) signaling pathway by removing lysine-63-linked polyubiquitin chains from NF-κB activating proteins. In order to investigate the role of CYLD in apoptotic signaling in human hepatocellular carcinoma (HCC) cells, we first studied the expression levels of CYLD in HCC tissues. CYLD expression was lower in HCC both at protein and mRNA levels compared to the surrounding non-malignant tissue. In order to further study the role of CYLD in the apoptotic sensitivity of HCC cells, CYLD was specifically down-regulated in HCC cell lines via RNA interference. The specific down-regulation of CYLD resulted in increased resistance towards treatment with doxorubicin, 5-fluorouracil and cisplatin. In addition, the down-regulation of CYLD in HCC cells decreased the sensitivity towards tumor necrosis factor-α-induced apoptosis. The CYLD knockdown also led to the degradation of the NF-κB inhibitor, IκB-α, resulting in enhanced NF-κB activity in HCC cells. Finally, we found that CYLD expression was triggered by the multikinase inhibitor, sorafenib, by the inhibition of Raf-1, as well as by the blockage of the pro-survival kinases, MEK (U0126) and the epidermal growth factor receptor (AG1478). In summary, we show that CYLD is down-regulated in human HCC and is involved in the apoptotic resistance of HCC cells. Our data identify the reconstitution of CYLD expression as an attractive approach for overcoming resistance to treatment in HCC.     

HA14-1 sensitizes TNF-α-induced apoptosis via inhibition of the NF-κB signaling pathway: Involvement of reactive oxygen species and JNK

Nuclear factor-kappa B (NF-κB) activation by tumor necrosis factor-alpha (TNF-α) attenuates the TNF-α-induced apoptosis pathway. Thus, blockage of NF-κB activity may improve the anti-cancer activity of TNF-α. HA14-1 induces apoptosis in various human cancer cells, and the molecular mechanisms of this action remain to be fully characterized. The present study evaluated the involvement of NF-κB, reactive oxygen species (ROS), and c-Jun N-terminal kinase (JNK) in the effects of HA14-1 by examining the sensitization effect on TNF-α-induced apoptosis in human leukemia cells. Such sensitization is closely associated with the inhibitory effect of HA14-1 on TNF-α-mediated NF-κB activation. HA14-1 suppressed NF-κB activation through inhibition of phosphorylation and degradation of IκBα. This inhibition was correlated with suppression of NF-κB-dependent gene products (c-myc, cyclin D1, cox-2, and IAP-1). Additionally, the present findings provide evidence of a critical role of ROS accumulation induced by HA14-1 in TNF-α-induced apoptosis. Moreover, HA14-1 also markedly sustained TNF-α-mediated JNK activation. A specific JNK inhibitor abolished the sensitization effect of HA14-1 on TNF-α-induced apoptosis. Taken together, these results indicate that ROS and JNK represent important signals in HA14-1 sensitization in TNF-α-induced apoptosis.     

倍半萜烯内酯对鼻咽癌细胞内核因子-кB活化的影响

背景与目的:探讨倍半萜烯内酯化合物对人鼻咽癌(nasopharyngeal carcinoma,NPC)细胞内核因子-κB(NF-κB)信号转导活化的影响。材料与方法:采用小白菊内酯(parthenolide,PN)作为受试物,以对PN诱导转归敏感的CNE1细胞给予TNF-α预诱导建立模型,PN处理后提取胞质和胞核蛋白,分别检测IκBα降解、NF-κB p65亚单位活化后核内迁移,电泳迁移率改变试验(EMSA)检测核内活化NF-κB的DNA结合活性。进行PN剂量和作用时间依赖关系分析。结果:阴性对照组IκBα蛋白存在于胞质中,PN处理组使TNF-α诱导的胞质IκBα蛋白降解被抑制、胞核内蛋白含量减少;相应地,阴性对照组p65亚单位在胞质中含量高于胞核内,PN处理组抑制TNF-α诱导的胞质p65核转位;同步进行的EMSA可见,PN处理组NF-κB核结合活性比TNF-α诱导组明显降低。随PN处理时间(0.5~4h)和剂量(5~25μmol/L)增加,胞质中IκBα蛋白降解的抑制作用增强(其蛋白含量增加),胞核内p65亚单位蛋白减少,EMSA结合活性降低,呈明显的剂量和时间依赖性(P均<0.05)。结论:PN可影响NPC细胞内NF-κB因子的活化,提示PN对TNF-α诱导NF-κB信号的抑制作用可能是PN诱导NPC细胞凋亡敏感性的分子机制之一。     

NF-κB inhibition in human hepatocellular carcinoma and its potential as adjunct to sorafenib based therapy

Nuclear factor-kappaB (NF-κB) has been shown to play an important role in the development and progression of cancer. In this study, we systematically examined NF-κBp65 signaling pathway in both human hepatocellular carcinoma (HCC) tissue and HCC cell lines. NF-κBp65 signaling pathway is aberrantly expressed and activated in both human HCC tissue and HCC Hep3B cells. Inhibition of NF-κB activity significantly reduced proliferation and invasion of Hep3B cells as well as down-regulated the expression of invasion-related molecules including matrix metalloproteinase (MMP)-2, MMP-9, membrane type-1 MMP (MT1-MMP), urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF). Hep3B cells exhibited a dose-dependent increase in apoptosis after receiving sorafenib treatment. Inhibition of NF-κB activity strongly sensitized Hep3B cells to sorafenib-induced cell death. Mechanistically, combined treatment of sorafenib and NF-κB inhibition enhanced inhibition of MAPK signaling and down-regulation of anti-apoptotic protein Mcl-1 expression. These observations indicate that inhibition of NF-κB may be a potential antineoplastic therapy for HCC, especially the combination of NF-κB inhibition and sorafenib provides a novel therapeutic strategy for patients with advanced-stage HCC.     

蜕皮激素对顺铂诱导小鼠肝损伤的保护作用及机制研究

目的:探讨蜕皮激素对顺铂导致小鼠肝损伤的保护作用及机制。方法:采用昆明小鼠和AML12细胞构建顺铂肝损伤体内及体外模型，应用蜕皮激素对顺铂组进行干预，通过HE染色观察肝脏组织病理形态，ELISA和荧光染色观测细胞内ROS的产生及细胞凋亡，RT-qPCR检测组织及细胞内氧化应激、炎症和凋亡相关生物标志物的表达。转录组测序测定其作用靶点，RT-qPCR和Western blot验证靶基因mRNA和蛋白表达。结果:与空白组相比，顺铂组小鼠肝细胞排列疏松，部分细胞轻度肿胀，凋亡细胞数量、血清内ALT、AST、肝细胞内ROS、MDA含量明显增多（P＜0.01），GSH-PX、SOD含量明显降低（P＜0.01），提示顺铂对肝功能造成明显损伤；与顺铂组相比，蜕皮激素组小鼠肝细胞损伤程度明显减轻，上述各项指标均有不同程度改善，表明其对肝功能具有保护作用。RT-qPCR结果显示，蜕皮激素组NF-κB、TNF-α、IL-1β、Caspase-3、Caspase-9、Hmox1、PI3K、Nqo1 mRNA相对表达量均明显降低，Nrf2 mRNA相对表达量明显升高（P＜0.01）。转录组测序及验证实验表明，蜕皮激素通过调控PDK1低表达，Dnmt3b、MEK5高表达，进而保护肝功能。结论:蜕皮激素能够通过炎症、氧化应激和凋亡抑制途径共同作用于肝细胞，并直接影响Dnmt3b、MEK5、PDK1的表达从而可能对顺铂导致的肝损伤具有保护作用。     

Inhibition of the tumour necrosis factor-alpha autocrine loop enhances the sensitivity of multiple myeloma cells to anticancer drugs

Several autocrine soluble factors, including macrophage inflammatory protein-1α and tumour necrosis factor-alpha (TNF-α), promote the survival and growth of multiple myeloma (MM) cells. We hypothesised that inhibition of the TNF-α autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, a TNF-α-neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of anticancer drugs on MM cells. In addition, combination treatment with the TNF-α-neutralizing antibody and the chemotherapy agent melphalan inhibited nuclear factor κB (NF-κB) p65 nuclear translocation and mammalian target of rapamycin (mTOR) activation and upregulated the expression of Bax and Bim. Treatment of ARH-77 cells with the NF-κB inhibitor dimethyl fumarate or the mTOR inhibitor rapamycin suppressed NF-κB p65 nuclear translocation and enhanced the cytotoxic effect of melphalan. Furthermore, infliximab, a monoclonal antibody against TNF-α, also enhanced the cytotoxic effect of anticancer drugs in ARH-77 cells. These results indicated that TNF-α-neutralizing antibodies or infliximab enhanced the cytotoxic effect of anticancer drugs by suppressing the TNF receptor/mTOR/NF-κB pathways. The inhibition of TNF-α may thus provide a new therapeutic approach to control tumour progression and bone destruction in MM patients.     

冻融治疗后肝癌库普弗细胞分泌功能的变化

Objective To explore the effect of Kupffer cells (KCs) secretion on HCC cells after cryoablation and its mechanism. Methods Sixteen groups were divided: 37°C control group, three hypothermia groups(0°C, 5°C, 10°C), freeze-thaw necrotic substances group, three combined stimulation group and 8 parallel groups for each group. In the 8 parallel groups, pyrrolidine dithiocarbamate(PDTC) was added, and the final concentration was 100 µmol/ml. The concentration of TNF-α, IL-1β and INF-γ in 16 groups of KCs supernatant were determined by Elisa method. The expression of NF-κB protein in each group was detected by Western blot. Results The secretion of inflammatory factors TNF-α, IL-1β and INF-γ increased after combined stimulation of KCs with hypothermia and freeze-thaw necrotic substances (P<0.01). The combined stimulation of low temperature (0°C, 5°C, 10°C) and freeze-thaw necrotic substances in the three groups showed superposition effect. Hypothermia stimulation had no significant effect on the expression of NF- κB protein (P>0.05), but the expression of NF-κB protein in the combined stimulation group and freezethaw necrosis substances group were significantly up-regulated (P<0.05). Treated with NF-κB inhibitor, the expression of NF-κB protein and the secretion level of inflammatory factors did not change significantly among 8 parallel groups (P>0.05). The concentrations of TNF-α, IL-1β and INF-γ were positively correlated with the expression of NF-κB protein in the culture medium of KCs (r≥0.870, P=0.000). Conclusion Freeze-thaw therapy could enhance the function of KCs secretion cytokines, and to a certain extent, it could induce inflammatory response and eliminate tumor cells. The secretion function of KCs may play a role through NF-κB signaling pathway. © 2020, CHINA RESEARCH ON PREVENTION AND TREATMENT. All rights reserved.     

煤焦沥青烟提取物对BEAS-2B细胞Nrf2及NQO1表达的影响

目的：研究煤焦沥青烟提取物对人肺支气管上皮细胞（BEAS-2B）Nrf2、NQO1 mRNA及Nrf2蛋白表达的影响。方法：煤焦沥青烟提取物以0．00、1．25、2．50、5．00、10．00μg／ml分别染毒BEAS-2B细胞24h后,提取细胞总RNA和总蛋白。采用RT-PCR检测Nrf2、NQO1 mRNA的相对表达量,Western blot测定Nrf2蛋白相对表达量。结果：染毒组BEAS-2B细胞Nrf2 mRNA及蛋白相对表达量均低于对照组,差别均有统计学意义（P均〈0．05）,在1．25—5．00μg／ml浓度范围内,随着染毒浓度的增加,Nrf2 mRNA和蛋白表达呈递增趋势,而当染毒浓度为10．00μg／ml时,Nrf2 mRNA和蛋白表达量均有所减少（P〈0．05）。随着染毒浓度的增加,NQO1基因表达水平升高,在5．00、10．00μg／ml时NQO1基因相对表达量比对照组高（P〈0．05）。结论：在煤焦沥青烟提取物浓度较低时（1．25～5．00μg／ml）,随染毒浓度增加Nrf2表达水平升高,可能上调NQO1基因的表达,增强BEAS-2B细胞氧化应激能力。     

P2X7R激动剂BzATP对非小细胞肺癌A549细胞生长和凋亡的影响

目的 研究配体门控离子通道P2X7受体(P2X7R)在非小细胞肺癌A549细胞中的表达,观察P2X7R激动剂2'-3'-O-(4-苯甲酰-苯甲酰)腺苷三磷酸三乙烷胺盐(BzATP)对A549细胞生长及凋亡的影响,并探究相关作用机制.方法 采用免疫荧光法检测P2X7R在A549细胞中的表达.用不同浓度的BzATP(150、300、600 μmol/L)处理,未用BzATP干预的细胞作为对照组.采用四甲基偶氮唑蓝(MTT)和Hoest33342染色法分别检测细胞存活率与凋亡情况,酶联免疫吸附试验检测上清液中肿瘤坏死因子-α(TNF-α)的浓度,Western blotting检测核转录因子-κB(NF-κB) p65、NF-κB抑制因子α(IκBα)及磷酸化NF-κB抑制因子α(phospho-IκBα)蛋白的表达.结果 P2X7R在A549细胞膜上表达.在300、600 μmol/L BzATP作用下A549细胞存活率分别为(67.87±8.98)％、(44.73±6.92)％,较对照组(98.60±1.44)％明显下降,差异均具有统计学意义(=4.481,P=0.027;t =3.920,P=0.038).BzATP可促进细胞凋亡,并且300、600 μmol/L BzATP可上调细胞培养上清中TNF-α浓度,分别为(57.35±6.41)pg/ml、(78.63±11.33) pg/ml,与对照组(42.56±0.37) pg/ml比较差异具有统计学意义(t=6.410,P=0.035;t=11.330,P=0.005).此外,BzATP可下调NF-κB p65的表达,上调IκBα的表达,对phospho-IκBα的表达无明显作用.结论 P2X7R表达于A549细胞膜,BzATP能够抑制细胞增殖,促进细胞凋亡,其作用机制可能与促进细胞中TNF-α的释放,抑制NF-κB通路有关.     

Luteolin Sensitizes Two Oxaliplatin-Resistant Colorectal Cancer Cell Lines to Chemotherapeutic Drugs Via Inhibition of the Nrf2 Pathway

Oxaliplatin is a first-line therapy for colorectal cancer, but cancer cell resistance to the drug compromisesits efficacy. To explore mechanisms of drug resistance, we treated colorectal cancer cells (HCT116 and SW620)long-term with oxaliplatin and established stable oxaliplatin-resistant lines (HCT116-OX and SW620-OX).Compared with parental cell lines, IC50s for various chemotherapeutic agents (oxaliplatin, cisplatin anddoxorubicin) were increased in oxaliplatin-resistant cell lines and this was accompanied by activation of nuclearfactor erythroid-2 p45-related factor 2 (Nrf2) and NADPH quinone oxidoreductase 1 (NQO1) . Furthermore,luteolin inhibited the Nrf2 pathway in oxaliplatin-resistant cell lines in a dose-dependent manner. Luteolin alsoinhibited Nrf2 target gene [NQO1, heme oxygenase-1 (HO-1) and GSTα1/2] expression and decreased reducedglutathione in wild type mouse small intestinal cells. There was no apparent effect in Nrf2-/- mice. Luteolincombined with other chemotherapeutics had greater anti-cancer activity in resistant cell lines (combined indexvalues below 1), indicating a synergistic effect. Therefore, adaptive activation of Nrf2 may contribute to thedevelopment of acquired drug-resistance and luteolin could restore sensitivity of oxaliplatin-resistant cell linesto chemotherapeutic drugs. Inhibition of the Nrf2 pathway may be the mechanism for this restored therapeuticresponse.