类泛素蛋白FAT10对人肾上皮细胞α-烯醇酶共价修饰的初步研究

目的:构建带Flag标签的α-烯醇酶(ENO1)基因以及带Myc标签的人白细胞抗原F相关转录物10(FAT10)基因的真核表达质粒载体,鉴定ENO1和FAT10在人肾上皮细胞株HEK293中的表达,检测类泛素蛋白FAT10是否共价修饰ENO1。方法:以人宫颈癌细胞系He La的c DNA为模板进行PCR,得到eno1目的片段,与分别经EcoRⅤ和SalⅠ酶切的pFlag-CMV载体连接得到重组质粒pFlag-CMV-eno1并进行鉴定测序;以人脑组织c DNA文库为模板进行PCR,得到fat10目的片段,与分别经Eco RⅠ和XhoⅠ酶切的p CMV-Myc载体连接得到重组质粒p CMV-Myc-fat10并进行鉴定并测序。将重组质粒用脂质体法转染HEK293细胞,用Western印迹检测ENO1和FAT10蛋白的表达。将重组质粒共转染HEK293细胞,用免疫共沉淀检测FAT10对ENO1的修饰情况。结果:重组克隆载体内的eno1和fat10序列与Gen Bank报告的序列完全一致;转染HEK293细胞后,ENO1和FAT10蛋白过表达;FAT10能够共价修饰ENO1。结论:类泛素蛋白FAT10共价修饰ENO1,为肿瘤的发生、发展及迁移的研究提供了新的研究思路。     

LIGHT及IFN-γ对MIN6细胞凋亡和Bax表达的影响

目的研究细胞因子LIGHT及IFN-γ对小鼠胰岛素瘤细胞株MIN6细胞凋亡及促凋亡基因Bax蛋白表达的影响。方法体外培养MIN6细胞,将细胞分为4组:(1)未处理组(细胞对照组);(2)LIGHT组;(3)IFN-γ组;(4)LIGHT+IFN-γ组,用LIGHT与IFN-γ分别单独或联合刺激MIN6细胞24 h。采用MTT法检测细胞存活率,激光共聚焦显微镜观察MIN6细胞核形态,流式细胞仪测定细胞凋亡率。利用Western blot分析基因Bax蛋白表达变化,将Bax(siRNA)转染入MIN6细胞,用LIGHT和IFN-γ刺激24 h,Western blot检测Bax蛋白表达,MTT法检测细胞存活率变化。结果 LIGHT或IFN-γ单独刺激仅能引起MIN6细胞存活率轻微下降,但LIGHT与IFN-γ联合刺激能显著降低细胞存活率(P0.05),细胞核可见凋亡小体,细胞凋亡率(28.80%)明显高于LIGHT或IFN-γ组(4.42%和6.70%),且Bax蛋白含量增加。Bax siRNA降低了MIN6细胞Bax基因的表达,Bax siRNA+LIGHT+IFN-γ组的细胞存活率显著高于LIGHT+IFN-γ组和阴性对照siRNA+LIGHT+IFN-γ组(P0.05)。结论 LIGHT与IFN-γ联合刺激能促进MIN6细胞凋亡,且与促凋亡基因Bax表达上调有关。     

快速高效检测植物体内蛋白泛素化修饰研究方法

UPS参与植物中绝大多数的信号转导通路。其中, 一些激素的受体本身就是E3泛素连接酶, 如茉莉酸(JA)受体COI1和生长素(auxin)受体TIR1都是F-box蛋白, 它们通过特异性介导相应转录抑制子的泛素化降解来传递激素信号, 但对于整个UPS体系而言, 由于技术的限制, 迄今为止仅见少量泛素连接酶与特异性底物间生化机制的报道。用大肠杆菌(Escherichia coli)表达蛋白实施泛素连接酶泛素化修饰底物的体外实验是验证泛素连接酶/底物对的常用方法, 但由于体外实验缺乏某些蛋白必需的转录后修饰, 导致实验结果有时存在假阴性。利用农杆菌注射烟草(Nicotiana benthamiana)瞬时表达蛋白的方法, 建立高效的植物体内检测蛋白泛素化系统, 可以快速检测蛋白泛素化, 包括检测泛素连接酶和底物的特异性相互作用、底物蛋白的自身泛素化、泛素连接酶对底物降解的促进作用、26S蛋白酶体抑制剂MG132对底物降解的抑制作用以及用植物内源表达蛋白进行体外泛素化反应。     

快速高效检测植物体内蛋白泛素化修饰研究方法

UPS参与植物中绝大多数的信号转导通路。其中, 一些激素的受体本身就是E3泛素连接酶, 如茉莉酸(JA)受体COI1和生长素(auxin)受体TIR1都是F-box蛋白, 它们通过特异性介导相应转录抑制子的泛素化降解来传递激素信号, 但对于整个UPS体系而言, 由于技术的限制, 迄今为止仅见少量泛素连接酶与特异性底物间生化机制的报道。用大肠杆菌(Escherichia coli)表达蛋白实施泛素连接酶泛素化修饰底物的体外实验是验证泛素连接酶/底物对的常用方法, 但由于体外实验缺乏某些蛋白必需的转录后修饰, 导致实验结果有时存在假阴性。利用农杆菌注射烟草(Nicotiana benthamiana)瞬时表达蛋白的方法, 建立高效的植物体内检测蛋白泛素化系统, 可以快速检测蛋白泛素化, 包括检测泛素连接酶和底物的特异性相互作用、底物蛋白的自身泛素化、泛素连接酶对底物降解的促进作用、26S蛋白酶体抑制剂MG132对底物降解的抑制作用以及用植物内源表达蛋白进行体外泛素化反应。     

HIF-1α的可逆性SUMO化修饰

低氧诱导因子1(hypoxia inducible factor-1, HIF-1)是参与调节机体氧平衡的重要转录因子,在细胞低氧应答反应中起核心作用,能调节100多种涉及低氧应激下细胞适应和存活的靶基因．HIF-1由氧敏感的α亚基和在细胞内稳定表达的β亚基组成．其中α亚基可受到多种翻译后化学修饰作用,如在常氧下,HIF-1α通过泛素化蛋白酶修饰并导致其快速降解．最近几年发现的泛素样蛋白家族成员小泛素蛋白样修饰蛋白（SUMO）也能与HIF-1α共价结合．SUMO是一种分子量约为12 kD的小蛋白,从拟南芥到人类普遍存在．SUMO可共价结合许多靶底物蛋白,并对其进行翻译后修饰,该过程称为SUMO化．与泛素化蛋白酶体途径不同的是,SUMO化修饰能在常氧和相对低氧的条件下调节HIF-1α蛋白的稳定性,从而改变其转录活性．SUMO化是一个可逆的动态过程,可被特异性蛋白酶ULP/SENP将其从底物上去除．本文主要就HIF-1α的可逆性SUMO化修饰作一综述．     

γ干扰素抑制幼儿支气管上皮细胞MUC5AC表达

本研究主要探讨了IFN-γ在黏蛋白产生中的作用,特别是在儿童支气管上皮细胞中的MUC5AC转录。通过采用人肺黏液表皮样癌细胞系(NCI-H292)和正常人支气管上皮细胞(NHBE),本研究评估了IFN-γ对MUC5AC转录的影响,发现转化生长因子(TGF)-α和双链RNA (polyI:C)诱导的MUC5AC mRNA和蛋白表达被IFN-γ以浓度依赖性方式抑制。IFN-γ对TGF-α和polyI:C诱导的表皮生长因子受体(EGFR)和细胞外信号调节激酶(ERK)的激活作用有限。染色质免疫沉淀实验表明Sp1与位于MUC5AC启动子上的同源序列结合。Sp1抑制剂光神霉素A抑制MUC5AC mRNA,表明Sp 1在MUC5AC诱导中起关键作用,同时IFN-γ阻碍Sp1与MUC5AC启动子的结合。本研究初步表明,IFN-γ可以抑制MUC5AC的表达,干扰Sp1与其靶序列的结合。     

辛伐他汀对IFN-γ 诱导的人外周血单个核细胞OX40L 表达的影响

目的:探讨经IFN-γ刺激后,人外周血单个核细胞OX40L表达的变化,以及辛伐他汀对单个核细胞OX40L表达的影响.方法:将实验标本随机分为2组,分别干扰素-γ(IFN-γ)刺激组、辛伐他汀干预组.应用RT-PCR及Western blotting技术,观察IFN-γ诱导的人外周血单个核细胞OX40L表达情况及辛伐他汀对人单个核细胞OX40L表达的影响.结果:1.1000U/mlIFN-与人单个核细胞共同培养24h后,OX40L mRNA和蛋白水平的表达明显增加.2.预先给予10 mol/L的辛伐他汀干预1h可以明显降低IFN-诱导的OX40L表达.结论:IFN-可诱导人单个核细胞OX40L表达.辛伐他汀可以抑制IFN-诱导人单个核细胞OX40L表达的增强,从而可能抑制了OX40L信号通路介导的与炎症有关的血管损伤,延缓动脉粥样硬化的进程.     

TNF-α在PMA和IFN-γ诱导U 937细胞生长和分化过程中的作用

本文报道了佛波酯(PMA)和γ-干扰素(IFN-γ)对U937细胞生长和分化的调控作用及其机制。PMA和IFN-γ能以剂量依赖的方式诱导U937细胞向成熟单核/巨噬细胞样细胞分化,同时抑制其细胞的生长。实验发现PMA和IFN-γ可诱导U937细胞表达TNF-α特异性mRNA和蛋白质。U937细胞培养中加入特异性抗TNF-α抗体可以抑制PMA和IFN-γ诱导U937细胞的分化和生长。这说明内源性的TNF-α在介导PMA和IFN-γ上述生物效应过程中起一定作用。TNF-α对U937细胞的这种调节作用与其胞毒作用机制不同     

5-氮杂胞苷对T淋巴细胞Jurkat miR-126及IFN-γ、GATA3、ROR-γ、Foxp3表达的影响

目的:探究甲基化酶抑制剂5-氮杂胞苷(5-azacytidin,5-aza)对T淋巴细胞(Jurkat)mi R-126、Th1/Th2、Th17/Treg细胞亚群及因子IFN-γ、GATA3、ROR-γ和Foxp3的调控作用。方法:采用不同浓度5-aza干预T淋巴细胞,24 h、48 h后检测其对细胞增殖抑制作用;实时荧光定量PCR、Western Blot检测5-aza干预后mi R-126表达水平以及IFN-γ、GATA3、ROR-γ和Foxp3的m RNA及蛋白表达水平;流式细胞术检测5-aza干预后Th1/Th2、Th17/Treg细胞亚群分化比例。结果:甲基化酶抑制剂干预T淋巴细胞后,细胞抑制率随5-aza浓度增大及作用时间延长呈递增趋势(P<0.01);细胞抑制率在24 h、48 h,低、中、高浓度下分别为(14.73±0.93)%、(32.67±8.40)%、(60.87±5.78)%以及(18.98±0.73)%、(39.80±8.42)%、(64.11±6.04)%;抑制率在24 h与48 h之间无差异(P>0.05)。甲基化酶抑制剂干预后mi R-126表达降低(P<0.01);IFN-γ蛋白表达降低(P<0.05)、Th1细胞亚群数目降低(P<0.01);GATA3 m RNA和蛋白表达升高(P<0.05)、Th2细胞亚群数目增加(P<0.01);ROR-γ蛋白表达降低(P>0.05)、Th17细胞亚群数目降低(P<0.05);Foxp3 m RNA和蛋白表达升高(P>0.05)、Treg细胞亚群数目增加(P<0.05)。结论:甲基化酶抑制剂可以下调Jurkat细胞mi R-126基因表达;下调Th1、Th17细胞亚群分化,上调Th2、Treg细胞亚群分化;调控细胞因子IFN-γ、GATA3、ROR-γ和Foxp3的表达。     

The ubiquitin-like modifier FAT10 in cancer development

During the last years it has emerged that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 expression is highly up-regulated by pro-inflammatory cytokines IFN-γ and TNF-α in all cell types and tissues and it was also found to be up-regulated in many cancer types such as glioma, colorectal, liver or gastric cancer. While pro-inflammatory cytokines within the tumor microenvironment probably contribute to FAT10 overexpression, an increasing body of evidence argues that pro-malignant capacities of FAT10 itself largely underlie its broad and intense overexpression in tumor tissues. FAT10 thereby regulates pathways involved in cancer development such as the NF-κB- or Wnt-signaling. Moreover, FAT10 directly interacts with and influences downstream targets such as MAD2, p53 or β-catenin, leading to enhanced survival, proliferation, invasion and metastasis formation of cancer cells but also of non-malignant cells. In this review we will provide an overview of the regulation of FAT10 expression as well as its function in carcinogenesis.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

香菇C91-3菌丝发酵蛋白对S180小鼠细胞免疫功能和IFN-γ及TNF-α的作用

目的观察香菇C91-3菌丝发酵蛋白对荷瘤小鼠免疫系统的作用。方法昆明小鼠24只,S180瘤细胞荷瘤建立动物模型,随机平均分为3组：香菇C91-3菌丝发酵蛋白治疗组、环磷酰胺治疗组、生理盐水对照组。观察香菇C91-3菌丝发酵蛋白对荷瘤小鼠NK细胞活性、淋巴细胞转化率、IFN-γ和TNF-α等免疫指标的影响。结果香菇C91-3菌丝发酵蛋白能够显著提高治疗组小鼠的NK细胞活性、淋巴细胞转化率和血清中IFN-γ和TNF-α的含量。结论香菇C91-3菌丝发酵蛋白能够显著提高荷瘤小鼠免疫系统的活性。     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.     

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.     

Effect of tumor necrosis factor-α and interferon-γ on the growth of a human salivary gland cell line

Interferon-γ (IFN-γ) is a product of activated T-lymphocytes, and tumor necrosis factor-α (TNF-α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-α and IFN-γ on a human submandibular gland epithelial cell line (HSG). IFN-γ caused a concentration-dependent decrease in HSG cell growth (～70% in 6 days). Conversely, TNF-α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-α and 1,000 units/ml of IFN-γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-γ alone and that of IFN-γ and TNF-α in combination were blocked completely using an antibody to the IFN-γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-γ, with or without TNF-α, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-α. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Activation of TOLLIP by porin prevents TLR2-associated IFN-γ and TNF-α-induced apoptosis of intestinal epithelial cells

Interferon (IFN)-γ and tumor necrosis factor (TNF)-α cause chronic inflammation of the intestine leading to progression of inflammatory bowel disease (IBD), which is manifested through rapid apoptosis of the intestinal epithelial cells (iECs). Here, we show inhibition of IFN-γ and TNF-α-induced apoptosis of INT-407 cells by porin, a microbe-associated molecular pattern (MAMP) with affinity for toll-like receptor (TLR)2 and commonly present in Gram-negative bacteria. Proinflammatory cytokines induce apoptosis by activation of caspase 8 that triggers caspase 9 through Bax finally leading to activation of caspase 3, the executioner caspase. Interestingly, while IFN-γ and TNF-α promotes Bax expression, in contrast porin up-regulates anti-apoptotic Bcl-xL resulting in iEC survivability. We show elevated expression of TLR2 is a key requisite for IFN-γ and TNF-α mediated caspase 8 up-regulation that contributes to apoptosis of iECs. Down-regulation of TLR2 expression is central for checking apoptosis which is achieved by elevated level of toll-interacting protein (TOLLIP) in presence of porin. Attempts to limit IBD is in progress with anti-IFN-γ and anti-TNF-α Abs or use of IL-10. Although probiotic bacterial proteins have shown to successfully reduce IFN-γ and TNF-α mediated apoptosis, the exact mechanism of their action has remained elusive. This study identifies the underlying sequential events of transient TLR2 stimulation followed by its blocking in response to the bacterial outer membrane protein, which advocates intervention at TLR-juncture is crucial for controlling IBD.     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the β-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic β-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it’s activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.