A Novobiocin Derivative,XN4, Inhibits the Proliferation of Chronic Myeloid Leukemia Cells by Inducing Oxidative DNA Damage

XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). The inhibition of proliferation of K562 and K562/G01 cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The mRNA levels of NADPH oxidase 1-5 (Nox1-5) genes were evaluated by qRT-PCR. The levels of extracellular reactive oxygen species (ROS), DNA damage, apoptosis, and cell cycle progression were examined by flow cytometry (FCM). Protein levels were analyzed by immunoblotting. XN4 significantly inhibited the proliferation of K562 and K562/G01 cells, with IC₅₀ values of 3.75±0.07 µM and 2.63±0.43 µM, respectively. XN4 significantly increased the levels of Nox4 and Nox5 mRNA, stimulating the generation of intracellular ROS, inducing DNA damage and activating ATM-γ-H2AX signaling, which increased the number of cells in the S and G2/M phase of the cell cycle. Subsequently, XN4 induced apoptotic cell death by activating caspase-3 and PARP. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine (NAC). Additionally, XN4 can induce apoptosis in progenitor/stem cells isolated from CML patients’ bone marrow. In conclusion, XN4-induced DNA damage and cell apoptosis in CML cells is mediated by the generation of ROS.     

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.     

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage,Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients

Introduction

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.

Methods

In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2'',7''-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.

Results

In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.

Conclusions

In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.     

Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch

Transforming growth factor-β (TGF-β) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-β induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-β caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-β-induced apoptosis. However, vNox4, but not TGF-β, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-β enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-β-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.     

DNA damage signaling induced by the G-quadruplex ligand 12459 is modulated by PPM1D/WIP1 phosphatase

The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR–mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest. Surprisingly, increasing concentrations of 12459 leading to cell apoptosis induced a mechanism that bypasses the DNA damage signaling and leads to the dephosphorylation of Chk1 and γ-H2AX. We identified the phosphatase Protein Phosphatase Magnesium dependent 1D/Wild-type P53-Induced Phosphatase (PPM1D/WIP1) as a factor responsible for this dephosphorylation. SiRNA-mediated depletion of PPM1D/WIP1 reactivates the DNA damage signaling by 12459. In addition, PPM1D/WIP1 is activated by reactive oxygen species (ROS) induced by 12459. ROS generated by 12459 are sufficient to trigger an early DNA damage in A549 cells when PPM1D/WIP1 is depleted. However, ROS inactivation by N-acetyl cysteine (NAC) treatment does not change the apoptotic response induced by 12459. Because PPM1D expression was recently reported to modulate the recruitment of DNA repair molecules, our data would suggest a cycle of futile protection against 12459, thus leading to a delayed mechanism of cell death.     

The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease

Kidney disease progression can be affected by Na⁺ abundance. A key regulator of Na⁺ homeostasis is the ubiquitin ligase NEDD4-2 and its deficiency leads to increased Na⁺ transport activity and salt-sensitive progressive kidney damage. However, the mechanisms responsible for high Na⁺ induced damage remain poorly understood. Here we show that a high Na⁺ diet compromised kidney function in Nedd4-2-deficient mice, indicative of progression toward end-stage renal disease. Injury was characterized by enhanced tubule dilation and extracellular matrix accumulation, together with sustained activation of both Wnt/β-catenin and TGF-β signaling. Nedd4-2 knockout in cortical collecting duct cells also activated these pathways and led to epithelial–mesenchymal transition. Furthermore, low dietary Na⁺ rescued kidney disease in Nedd4-2-deficient mice and silenced Wnt/β-catenin and TGF-β signaling. Our study reveals the important role of NEDD4-2-dependent ubiquitination in Na⁺ homeostasis and protecting against aberrant Wnt/β-catenin/TGF-β signaling in progressive kidney disease.Subject terms: Stress signalling, End-stage renal disease     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.     

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3′-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII_170–755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.     

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.     

Release of cAMP Gating by the α6β4 Integrin Stimulates Lamellae Formation and the Chemotactic Migration of Invasive Carcinoma Cells

The α6β4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949–960). We demonstrate here using MDA-MB-435 breast carcinoma cells that α6β4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by α6β4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon α6β4 expression. Both lamellae formation and chemotactic migration are inhibited or “gated” by cAMP and our results reveal that a critical function of α6β4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that α6β4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.     

An Undesired Effect of Chemotherapy: GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT,NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4*

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.     

Xanthine Oxidase Mediates Hypoxia-Inducible Factor-2α Degradation by Intermittent Hypoxia

Sleep-disordered breathing with recurrent apnea produces chronic intermittent hypoxia (IH). We previously reported that IH leads to down-regulation of HIF-2α protein via a calpain-dependent signaling pathway resulting in oxidative stress. In the present study, we delineated the signaling pathways associated with calpain-dependent HIF-2α degradation in cell cultures and rats subjected to chronic IH. Reactive oxygen species (ROS) scavengers prevented HIF-2α degradation by IH and ROS mimetic decreased HIF-2α protein levels in rat pheochromocytoma PC12 cell cultures, suggesting that ROS mediate IH-induced HIF-2α degradation. IH activated xanthine oxidase (XO) by increased proteolytic conversion of xanthine dehydrogenase to XO. ROS generated by XO activated calpains, which contributed to HIF-2α degradation by IH. Calpain-induced HIF-2α degradation involves C-terminus but not the N-terminus of the HIF-2α protein. Pharmacological blockade as well as genetic knock down of XO prevented IH induced calpain activation and HIF-2α degradation in PC12 cells. Systemic administration of allopurinol to rats prevented IH-induced hypertension, oxidative stress and XO activation in adrenal medulla. These results demonstrate that ROS generated by XO activation mediates IH-induced HIF-2α degradation via activation of calpains.