BNIP3 deletion ameliorated enterovirus 71 infection-induced hand,foot and mouth disease via inhibiting apoptosis,autophagy, and inflammation in mice

Bcl2/adenovirus E1B protein-interacting protein 3 (BNIP3) plays a key role in cellular response to stress by regulating apoptosis and selective autophagy. The present study aimed to determine the effects of BNIP3 on enterovirus (EV) 71 infection-induced hand, foot and mouth disease (HFMD), and the apoptosis, autophagy and inflammatory in mice and SH-SY5Y human neuroblastoma cell line. Neonatal BALB/c mice were injected with EV 71 strain to induce the HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The BNIP3 mRNA and protein levels in the brain were increased in EV 71-infected mice. By contrast, the BNIP3-knockout (KO) mice with EV 71 infection had higher health score and survival rate. BNIP3 deletion reversed the increase of cleaved-caspase 3, cleaved-caspase 8, Bax, LC3 II and LC3 II/LC3 I levels, and the decrease of Bcl2 and Bcl2/Bax and LC3 I levels in the brain of mice with EV 71 infection. The EV 71 infection-induced increase of tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1β, IL-6, interferon (IFN)-α and IFN-γ levels were inhibited in BNIP3-KO mice. BNIP3 knockdown with small interfering RNA (siRNA) inhibited the EV 71 infection-induced the increases of apoptosis, autophagy and inflammatory factors in SH-SY5Y cells. BNIP3 overexpression further facilitated the EV 71 infection-induced increase of these inflammatory factors in SH-SY5Y cells. These results demonstrated that BNIP3 deletion ameliorated EV 71 infection-induced HFMD via inhibiting apoptosis, autophagy and inflammation in mice. BNIP3 may be a therapeutic target for HFMD.     

Variation in the kinetics of caspase-3 activation, Bcl-2 phosphorylation and apoptotic morphology in unselected human ovarian cancer cell lines as a response to docetaxel

Paclitaxel is able to cause cell death through the induction of apoptosis. Cell death characteristics for docetaxel have not yet been described in detail. We investigated four unselected human ovarian cancer cell lines for the sensitivity to a 1hr exposure to docetaxel and calculated the concentrations inhibiting 50% (IC(50)) and 90% (IC(90)) of cell growth. Of the cell lines A2780, H134, IGROV-1 (all wild-type p53) and OVCAR-3 (mutant, mt p53) A2780 was most sensitive and OVCAR-3 least sensitive. Equitoxic drug concentrations representing IC(90) values (25-510nM) were applied for 1hr to measure cell cycle distribution, DNA degradation, and to count apoptotic cell bodies and cells with multifragmented nuclei at various time-points after drug exposure. H134, IGROV-1 and OVCAR-3 showed a continued mitotic block up to at least 72hr and prolonged presence of cells with multifragmented nuclei. High percentages of apoptosis were calculated at 48hr and at later time-points. In contrast, A2780 cells accumulated in the S-phase of the cell cycle and apoptosis was hardly present. The changes in the expression levels of p53, p21/WAF1, Bax and Bcl-2, were not predictive for docetaxel-induced apoptosis. Caspase-3 activation occurred only in cells with accumulation in the G2/M phase starting as early as 8hr in OVCAR-3. Prolonged Bcl-2 phosphorylation was evident in OVCAR-3, visible at 24hr in H134 and IGROV-1, while this phenomenon did not occur in A2780. The mitogen-activated protein kinase pathway (JNKs/SAPKs or c-Jun N-terminal kinases/stress-activated protein kinases, JNK1/2; extracellular response kinase, ERK1/2; p38) did not seem to be directly involved in Bcl-2 phosphorylation or apoptosis. We conclude that docetaxel is able to activate caspase-3, induce Bcl-2 phosphorylation and apoptosis in cells that show a prolonged G2/M arrest, but cells may also die by a caspase-3-independent cell death mechanism.     

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations--global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36 B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.     

Impairment of autophagy: From hereditary disorder to drug intoxication

At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication.     

M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe

Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex.     

基于Bcl-2蛋白P2、P3活性区域抑制剂的设计、合成及体外活性研究

目的 探索Bcl-2蛋白P2、P3活性区域的构效关系,并设计合成活性较好的抑制剂.方法 基于对Bcl-2蛋白抑制剂、Bcl-2蛋白P2、P3活性区域的分析,利用Autodock 4.2软件设计对接,合成一系列新型Bcl-2小分子抑制剂;采用MTT法测定所合成的抑制剂对人恶性黑色素瘤细胞A375、人肝癌细胞HepG-2、人非小细胞肺癌细胞A549的体外抗肿瘤活性.结果 合成了13个新型Bcl-2小分子抑制剂(D1～ D13),其中,化合物D2、D6、D8的活性相对较好,对人恶性黑色素瘤细胞A375的抑制作用与阳性对照紫杉醇相当;化合物D1、D3、D4、D5也对A375细胞具一定的抑制活性.结论 获得了P2、P3活性区域的构效关系,有助于Bcl-2蛋白抑制剂的进一步研究.     

Apoptosis induction of 2'-hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells

2'-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2'-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78 kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.     

mGluR2/3 agonist LY379268, by enhancing the production of GDNF, induces a time-related phosphorylation of RET receptor and intracellular signaling Erk1/2 in mouse striatum

In the present study we aimed to verify if the enhancement of glial cell line-derived neurotrophic factor (GDNF) production in mouse striatum following treatment with LY379268 may also induce in the nigrostriatal system a time-related activation of RET receptor and its specific intracellular signaling. For this purpose, we have investigated the effects of LY379268 treatment on RET phosphorylation at the Tyr1062 and on downstream signaling Erk1/2, Akt and PLCγ1 pathway activation. The results showed that treatment with LY379268 (3 mg/kg) induces a significant increase of GDNF levels and time-related RET and Erk1/2 phosphorylation in the striatum. These increases were detected at 24 h and 48 h following LY379268 treatment. No changes were observed in the Akt and PLCγ1 phosphorylation levels. Similar results for p-Erk1/2 were observed in the substantia nigra. A complete block of LY379268 effect on striatal RET and p-Erk1/2 phosphorylation was observed in mice intrastriatal injected with anti-GDNF antibodies, suggesting a correlation between GDNF upregulation and RET activation. Overall, with present data we have shown that activation of mGluR2/3 receptors by LY379268 may be particularly promising for nigrostriatal dopaminergic system protection by enhancing striatal levels of GDNF/RET trophic system activity.     

14-3-3zeta interacts with human thromboxane receptors and is involved in the agonist-induced activation of the extracellular-signal-regulated kinase

Thromboxane receptor (TP) signaling results in a broad range of cellular responses including kinase activation and subsequent nuclear signaling events involved in cell transformation, proliferation, and cell survival. Proteins that may participate in the early signaling following receptor activation remain to be identified. We found that 14-3-3zeta is a novel protein interacting with TP intracellular loop 3 (i3) by yeast two-hybrid system. This interaction was further confirmed by GST pull-down and co-immunoprecipitation methods. Site-directed mutagenesis studies indicated that Pro-236 of the TP-i3 was involved in the binding to the 14-3-3zeta. Co-immunoprecipitation studies in the same cell lysate by TP antibody showed that TP binds not only with the 14-3-3zeta but also with the Raf-1. Our data also demonstrated that TP receptor activation induced by agonist rapidly recruited 14-3-3zeta and Raf-1 to form a complex with the TP on the plasma membrane. The significance of assembling this protein complex was examined by TP agonist-induced extracellular-signal-regulated kinase (ERK) phosphorylation in intact cells. TP agonist, I-BOP, induced ERK phosphorylation in HEK 293 cells expressing wild type TPalpha but significantly lower in those expressing TPalpha-P236V mutant. Attenuation of the expression of 14-3-3zeta by 14-3-3zeta siRNA decreased I-BOP-induced ERK phosphorylation indicating the involvement of the 14-3-3zeta in the signal transduction process. These results suggest that 14-3-3zeta may serve as a scaffold protein to form a protein complex consisting of TP, 14-3-3zeta, and Raf-1, and that this protein complex may be involved in the activation of ERK pathway following TP receptor activation.     

Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

Knockdown of NAPA using short-hairpin RNA sensitizes cancer cells to cisplatin: Implications to overcome chemoresistance

Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA—a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking—protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-α and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.     

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2+)-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H(2)O(2) (200 microM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.     

Inhibition of cathepsin S induces autophagy and apoptosis in human glioblastoma cell lines through ROS-mediated PI3K/AKT/mTOR/p70S6K and JNK signaling pathways

Cathepsin S is a lysosomal cysteine protease that is overexpressed in various cancer models and plays important role in tumorigenesis, however the mechanisms are unclear. In the present study, we found that inhibition of cathepsin S induced autophagy and mitochondrial apoptosis in human glioblastoma cells. Blockade of autophagy by either a chemical inhibitor or RNA interference attenuated cathespin S inhibition-induced apoptosis. Furthermore, autophagy and apoptosis induction was dependent on the suppression of phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) signaling pathway and activation of c-Jun N-terminal kinase (JNK) signaling pathway. In addition, reactive oxygen species (ROS) served as an upstream of PI3K/AKT/mTOR/p70S6K and JNK signaling pathways. In conclusion, the current study revealed that cathepsin S played an important role in the regulation of autophagy and apoptosis in human glioblastoma cells.