Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。     

c-Myc influences olaquindox-induced apoptosis in human hepatoma G2 cells

Olaquindox, a synthetic antimicrobial compound, was banned as feed additives in the U.S. and the EU. In China, the use of olaquindox is banned in poultry and aquaculture feed, restricted in livestock feed for growth promotion. Olaquindox's safety is the object of increasing attention. The present study was undertaken to investigate whether and how olaquindox elevates expression of c-Myc, which influences olaquindox-induced apoptosis in HepG2 cells. For a better understanding of c-Myc's role in susceptibility of human hepatoma G2 cells to olaquindox-induced apoptosis, two vectors (the pSilencer-cmyc(Si-cmyc) and the control vector) were transfected to HepG2 cells. The cells were pretreated with Si-cmyc, which expressed only 35-65% c-Myc protein levels compared to those of the parental cells and the control cells. We examined effects of olaquindox on reactive oxygen species (ROS) production in these c-Myc low-expressing cells, and on apoptosis. Our data revealed that ROS production induced by olaquindox treatment was partially blocked by Si-cmyc transfection and partly inhibited olaquindox-induced apoptosis through decreased ROS generation. Further data showed that olaquindox induced decreased ROS by Si-cmyc transfection through decreased cytochrome c release to cytosol, which inhibited apoptosis of the cells. These results suggest that c-Myc might be important during olaquindox-induced apoptosis in human hepatoma G2 cells.     

Role of tumor necrosis factor in preovulatory follicles of swine

The effects of tumor necrosis factor (TNF) on cultured porcine granulosa cells that were obtained from preovulatory follicles were studied with regard to following parameters: 1) TNF receptor type I expression, 2) progesterone receptor and transforming growth factor beta receptor type II (TbetaR II) as markers of luteinization, 3) proliferation, and 4) apoptosis. For comparative purposes the effects of TNF were also studied on insulin/forskolin-treated cells, as this treatment is well established to induce luteinization. Cytochemical methods followed by semiquantitative analysis were used. Our data show that TNF treatment upregulates TNF receptor type I expression in granulosa cells. TNF downregulates the expression of TbetaR II of insulin/forskolin-stimulated and of unstimulated cells. The progesterone receptor is also downregulated by the cytokine after insulin/forskolin-induced luteinization. Supplementation of the medium with TNF leads to increased proliferation and at the same time it induces apoptosis. Our results indicate that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis).     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Tumor necrosis factor induces apoptosis in hepatoma cells by increasing Ca(2+) release from the endoplasmic reticulum and suppressing Bcl-2 expression

Tumor necrosis factor (TNF) plays an import role in the control of apoptosis. The most well known apoptotic pathway regulated by TNF involves the TNFR1-associated death domain protein, Fas-associated death domain protein, and caspase-8. This study examines the mechanism of TNF-induced apoptosis in FaO rat hepatoma cells. TNF treatment significantly increased the percentage of apoptotic cells. TNF did not activate caspase-8 but activated caspase-3, -10, and -12. The effect of TNF on the expression of different members of the Bcl-2 family in these cells was studied. We observed no detectable changes in the steady-state levels of Bcl-X(L), Bax, and Bid, although TNF suppresses Bcl-2 expression. Dantrolene suppressed the inhibitory effect of TNF on Bcl-2 expression. TNF induced release of Ca(2+) from the endoplasmic reticulum (ER) that was blocked by dantrolene. Importantly, the expression of Bcl-2 blocked TNF-induced apoptosis and decreased TNF-induced Ca(2+) release. These results suggest that TNF induces apoptosis by a mechanism that involves increasing Ca(2+) release from the ER and suppression of Bcl-2 expression.     

Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.     

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti- versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by mu-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1-3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.     

Interleukin 1 and tumor necrosis factor stimulate cGMP formation in rat renal mesangial cells

Treatment of mesangial cells with recombinant human interleukin 1 beta (IL-1 beta) or recombinant human tumor necrosis factor alpha (TNF alpha) dose-dependently increased cGMP formation. Both IL-1 beta and TNF alpha-stimulated formation of cGMP occurred after a initial lag period of 4 to 8 hours. Treatment of cells with actinomycin D, cycloheximide or dexamethason completely abolished cytokine-induced cGMP formation. Furthermore, the guanylate cyclase inhibitor Methylene blue completely blocked IL-1 beta- and TNF alpha-stimulated cGMP generation. NG-mono-methyl-L-arginine attenuated IL-1 beta- and TNF alpha-induced cGMP production, an effect that was reversed by L-arginine.     

Inhibition of JNK by cellular stress- and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor alpha (TNFalpha) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNF alpha-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNF alpha-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates I kappa B kinase alpha. The phosphorylation of I kappa B kinase alpha and activation of NF kappa B mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNF alpha-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFalpha -induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NF kappa B pathway.     

Angiotensin II-induced mesangial cell apoptosis: role of oxidative stress

BACKGROUND: Angiotensin II (ANG II) has been shown to play a role in the induction of glomerular injury. In the present study, we evaluated the effects of ANG II on mesangial cell apoptosis and the involved molecular mechanism. MATERIALS AND METHODS: The effect of ANG II on apoptosis of mouse mesangial cells (MC) was evaluated by morphologic, DNA fragmentation and TUNEL assays. To evaluate the role of oxidative stress and involved mechanisms, we studied the effect of antioxidants, anti-TGF-beta antibody, inhibitors of nitric oxide synthase and modulators of cytosolic calcium/heme oxygenase (HO) activity. In addition, we studied the effect of ANG II on the generation of reactive oxygen species (ROS) by MCs. RESULTS: ANG II promoted apoptosis of MCs in a dose dependent manner. This effect of ANG II was not only associated with ROS production, but also inhibited by antioxidants. Both Anti-TGF-beta antibody and propranolol inhibited ANG II-induced ROS generation and apoptosis. BAPTA inhibited both ANG II- and TGF-beta-induced apoptosis. On the other hand, thapsigargin stimulated MC apoptosis under basal as well as ANG II/TGF-beta stimulated states. ANG II receptor types 1 and 2 antagonists attenuated the proapoptotic effect of ANG II. Hemin inhibited but zinc protoporphyrin enhanced the proapoptotic effect of ANG II. Propranolol increased HO activity; whereas pre-treatment with propranolol prevented ANG II-induced apoptosis. CONCLUSIONS: ANG II promotes MC apoptosis. This effect of ANG II is mediated through downstream signaling involving TGF-beta, phospholipase D, and Ca(2+), contributing to the activation of NADPH oxidase and generation of ROS. HO activity plays a modulatory role in ANG II- induced MC apoptosis.     

SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for this function and TNF-induced cathepsin G is a candidate target.

The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNF alpha-mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNF alpha-induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNF alpha but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNF alpha. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.     

Mechanisms of free-radical induction in relation to fenretinide-induced apoptosis of neuroblastoma

The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro l-argenine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A(2) or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.     

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.