IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.     

Lack of X-linked inhibitor of apoptosis protein leads to increased apoptosis and tissue loss following neonatal brain injury

Neurological deficits caused by H-I (hypoxia-ischaemia) to the perinatal brain are often severely debilitating and lead to motor impairment, intellectual disability and seizures. Perinatal brain injury is distinct from adult brain injury in that the developing brain is undergoing the normal process of neuronal elimination by apoptotic cell death and thus the apoptotic machinery is more easily engaged and activated in response to injury. Thus cell death in response to neonatal H-I brain injury is partially due to mitochondrial dysfunction and activation of the apoptosome and caspase 3. An important regulator of the apoptotic response following mitochondrial dysfunction is XIAP (X-linked inhibitor of apoptosis protein). XIAP inhibits apoptosis at the level of caspase 9 and caspase 3 activation, and lack of XIAP in vitro has been shown to lead to increased apoptotic cell death. In the present study we show that mice lacking the gene encoding the XIAP protein have an exacerbated response to neonatal H-I injury as measured by tissue loss at 7 days following the injury. In addition, when the XIAP-deficient mice were studied at 24 h post-H-I we found that the increase in injury correlates with an increased apoptotic response in the XIAP-deficient mice and also with brain imaging changes in T2-weighted magnetic resonance imaging and apparent diffusion coefficient that correspond to the location of apoptotic cell death. These results identify a critical role of XIAP in regulating neuronal apoptosis in vivo and demonstrate the enhanced vulnerability of neurons to injury in the absence of XIAP in the developing brain.     

Caspase-independent apoptosis is activated by diazepam-induced mitotic failure in HeLa cells,but not in human primary fibroblasts

DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.     

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue. This work was supported by a grant from the Wellcome Trust GR071469 (GIO) and the Chilean national science grants FONDECYT 1060495 (GIO) and 1020675 (MC). An erratum to this article is available at .     

Oxytocin increases invasive properties of endometrial cancer cells through phosphatidylinositol 3-kinase/AKT-dependent up-regulation of cyclooxygenase-1, -2, and X-linked inhibitor of apoptosis protein

Traditionally, oxytocin (OT) is well known to play a crucial role in the regulation of cyclic changes in the uterus, implantation of the embryo, and parturition. Recently, an additional role for OT has been identified in several types of cancer cells in which OT acts as a growth regulator. In endometrial cancer cells, OT is known to efficiently inhibit cellular proliferation. In the present study, we show that OT increases invasiveness of human endometrial carcinoma (HEC) cells, which are otherwise resistant to the growth-inhibiting effects of OT. Using pharmacological inhibitors, invasion assay, RNA interference, and immunofluorescence, we found that OT enhances the invasive properties of HEC cells through up-regulation of X-linked inhibitor of apoptosis protein (XIAP), matrix-metalloproteinase 2 (MMP2), and matrix-metalloproteinase 14 (MMP14). In addition, we show that OT-mediated invasion is both cyclooxygenase 1 (PTGS1) and cyclooxygenase-2 (PTGS2) dependent via the phosphatidylinositol 3-kinase/AKT (PIK3/AKT) pathway. PTGS2 knockdown by shRNA resulted in XIAP down-regulation. We also show that OT receptor is overexpressed in grade I to III endometrial cancer. Taken together, our results describe for the first time a novel role for OT in endometrial cancer cell invasion.     

Rapamycin antagonizes NF-kappaB nuclear translocation activated by TNF-alpha in primary vascular smooth muscle cells and enhances apoptosis

Several lines of evidence support the view that rapamycin inhibits NF-kappaB. TNF-alpha, a potent inducer of NF-kappaB, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-kappaB activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-alpha. Using EMSA, we found that TNF-alpha caused NF-kappaB nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited IkappaBalpha degradation, thereby preventing nuclear translocation. Activation of NF-kappaB was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-alpha-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-alpha was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented IkappaBalpha degradation nor increased apoptosis of cells incubated with TNF-alpha. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-alpha-induced NF-kappaB activation in VSMCs. Our findings show that rapamycin inhibits NF-kappaB activation and acts in concert with TNF-alpha in induction of VSMC apoptosis.     

Reduction of the antiapoptotic protein cFLIP enhances the susceptibility of human renal cancer cells to TRAIL apoptosis

Human renal carcinoma cells (RCCs) were sensitized to the apoptotic effects of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), by treatment with cycloheximide (CHX). In contrast to a previous study, a rapid and dramatic decrease in levels of cellular FLICE (Fas-associated death domain–like IL-1–converting enzyme) inhibitory protein (cFLIP) following cycloheximide treatment was observed in all RCCs studied. The unambiguous detection of this decrease in cFLIP was dependent on the quality of the particular antibody preparation used to detect cFLIP. Cycloheximide treatment caused no major change in levels of pro-caspase-8 or cell surface expression of TRAIL receptors. Therefore, cycloheximide treatment resulted in an increase in the pro-caspase-8 to cFLIP ratio, which correlated with sensitization to TRAIL-mediated apoptosis. Furthermore, treatment of human RCCs with small interfering oligoribonucleotides (siRNA) for cFLIP caused a reduction of cFLIP protein and sensitized cells to TRAIL-mediated apoptosis. We concluded that in the presence of an intact TRAIL signaling pathway, a significant reduction of cFLIP alone is sufficient to sensitize human RCCs to TRAIL apoptosis.The publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.     

Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to γ radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to γ radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types

Resistance of normal cells to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis is believed to be mediated by expression of two decoy receptors. Here we show that the expression and localisation of TRAIL receptors (TRAIL-Rs) vary between different cells and that resistance to TRAIL is mediated by different mechanisms. The decoy receptor, TRAIL-R3, appeared important in protection of endothelial cells, whereas lack of surface death receptor expression and as yet unknown intracellular inhibitor(s) of apoptosis downstream of caspase-3 may play a major role in protection of melanocytes and fibroblasts from TRAIL induced apoptosis, respectively. Differential subcellular location of decoy receptors may be an important determinant of their effectiveness in different types of normal cells.     

X-linked inhibitor of apoptosis protein mediates neddylation by itself but does not function as a NEDD8-E3 ligase for caspase-7

X-linked inhibitor of apoptosis protein (XIAP) is a potent antagonist of caspases, and functions as a ubiquitin-E3 ligase by itself and for caspases. Recently, NEDD8, a ubiquitin-like modifier, has been suggested to be used for modification of caspase-7 mediated by XIAP. However, it is not clear whether caspase-7 is a bona fide target for NEDD8. Here we showed that no neddylation of caspase-7 but that of XIAP itself was observed under the conditions in which caspase-7 was modified with ubiquitin. These results reveal that XIAP does not function as a NEDD8-E3 ligase for caspase-7 in vivo.     

Suppression of anoikis by v-Src but not by activated c-H-ras in human gallbladder epithelial cells

Detachment of anchorage-dependent normal epithelial cells from their substratum causes the type of apoptosis known as anoikis, whereas malignant cells can proliferate independently of anchorage. Because src and ras oncogenes are activated in many human cancers, we investigated their role and downstream signaling pathways in anoikis resistance, using HAG-1 human epithelial cells transfected with v-src or activated H-ras. Consequently, anchorage-dependent mock- or ras-transfected cells underwent anoikis. In contrast, anchorage-independent v-Src-transformed cells did not exhibit such apoptotic features. Focal adhesion kinase (FAK), a transducer of integrin, was only activated in v-Src-transformed cells. Herbimycin A, an Src kinase inhibitor, reduced tyrosyl phosphorylation of FAK and reversed resistance to anoikis. However, both protein kinase C (PKC) and phophatidylinositol-3 (PI-3) kinase inhibitors failed to induce anoikis. These data suggest that the ability of activated Src to prevent anoikis may be mediated by Src to a downstream signaling pathway involving FAK, but not Ras, PI-3 kinase, or PKC.     

Novel Polypyridyl chelators deplete cellular zinc and destabilize the X-linked inhibitor of apoptosis protein (XIAP) prior to induction of apoptosis in human prostate and breast cancer cells

X-linked inhibitor of apoptosis protein (XIAP), inhibits the initiation and execution phases of the apoptotic pathway. XIAP is the most potent member of the inhibitor of apoptosis protein (IAP) family of the endogenous caspase inhibitors. Therefore, targeting XIAP may be a promising strategy for the treatment of apoptosis-resistant malignancies. In this study, we systematically studied the relationships of chemical structures of several novel ligands to their zinc (Zn)-binding ability, molecular target XIAP, and tumor cell death-inducing activity. We show that treatment of PC-3 prostate cancer and MDA-MB-231 breast cancer cells with these membrane-permeable Zn-chelators with different Zn affinities results in varying degrees of XIAP depletion. Following decreased level of XIAP expression, we also show apoptosis-related caspase activation and cellular morphological changes upon treatment with strong Zn-chelators N4Py and BnTPEN. Addition of Zn has a full protective effect on the cells treated with these chelators, while iron (Fe) addition has only partial protection that, however, can be further increased to a comparable level of protection as Zn by inhibition of ROS generation, indicating that cell death effects mediated by Fe- but not Zn-complexes involve redox cycling. These findings suggest that strong Zn-chelating agents may be useful in the treatment of apoptosis-resistant human cancers.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.