Betulinic Acid Inhibits the Stemness of Gastric Cancer Cells by Regulating the GRP78-TGF-ß1 Signaling Pathway and Macrophage Polarization.

Cancer stemness is the process by which cancer cells acquire chemoresistance and self-renewal in the tumor microenvironment. Glucose-regulated protein 78 (GRP78) is a biomarker for gastric cancer and is involved in cancer stemness. By inducing cancer stemness in various types of cancer, the polarization of macrophages into tumor-associated macrophages (TAMs) controls tumor progression. Betulinic acid (BA) is a bioactive natural compound with anticancer properties. However, whether GRP78 regulates TAM-mediated cancer stemness in the tumor microenvironment and whether BA inhibits GRP78-mediated cancer stemness in gastric cancer remain unknown. In this study, we investigated the role of GRP78 in gastric cancer stemness in a tumor microenvironment regulated by BA. The results indicated that BA inhibited not only GRP78-mediated stemness-related protein expression and GRP78-TGF-ß-mediated macrophage polarization into TAMs, but also TAM-mediated cancer stemness. Therefore, BA is a promising candidate for clinical application in combination-chemotherapy targeting cancer stemness.

Optimization of Intraoperative Neural Monitoring of the Recurrent Laryngeal Nerve in Thyroid Surgery.

The application of intraoperative neural monitoring (IONM) has been widely accepted to improve surgical outcomes after thyroid surgery. The malfunction of an IONM system might interfere with surgical procedures. Thus, the development of anesthesia modalities aimed at ensuring functional neuromonitoring is essential. Two key issues should be taken into consideration for anesthetic management. Firstly, most patients undergo recurrent laryngeal nerve monitoring via surface electrodes embedded in an endotracheal tube. Thus, advanced video-assisted devices might optimize surface electrode positioning for improved neuromonitoring signaling accuracy. Secondly, neuromuscular blocking agents are routinely used during thyroid surgery. The ideal neuromuscular block should be deep enough for surgical relaxation at excision and recovered enough for an adequate signal f nerve stimulation. Proper neuromuscular block management could be achieved by titration doses of muscle relaxants and reversal agents.

IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of Gamma Interferon via Signaling Attenuation.

UNLABELLED: Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms. IMPORTANCE: Immunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.

Detection of reactive oxygen species during the cell cycle under normal culture conditions using a modified fixed-sample staining method.

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

An increase in integrin-linked kinase non-canonically confers NF-κB-mediated growth advantages to gastric cancer cells by activating ERK1/2.

BACKGROUND: Increased activity or expression of integrin-linked kinase (ILK), which regulates cell adhesion, migration, and proliferation, leads to oncogenesis. We identified the molecular basis for the regulation of ILK and its alternative role in conferring ERK1/2/NF-κB-mediated growth advantages to gastric cancer cells. RESULTS: Inhibiting ILK with short hairpin RNA or T315, a putative ILK inhibitor, abolished NF-κB-mediated the growth in the human gastric cancer cells AGS, SNU-1, MKN45, and GES-1. ILK stimulated Ras activity to activate the c-Raf/MEK1/2/ERK1/2/ribosomal S6 kinase/inhibitor of κBα/NF-κB signaling by facilitating the formation of the IQ motif-containing GTPase-activating protein 1 (IQGAP1)-Ras complex. Forced enzymatic ILK expression promoted cell growth by facilitating ERK1/2/NF-κB signaling. PI3K activation or decreased PTEN expression prolonged ERK1/2 activation by protecting ILK from proteasome-mediated degradation. C-terminus of heat shock cognate 70 interacting protein, an HSP90-associated E3 ubiquitin ligase, mediated ILK ubiquitination to control PI3K- and HSP90-regulated ILK stabilization and signaling. In addition to cell growth, the identified pathway promoted cell migration and reduced the sensitivity of gastric cancer cells to the anticancer agents 5-fluorouracil and cisplatin. Additionally, exogenous administration of EGF as well as overexpression of EGFR triggered ILK- and IQGAP1-regulated ERK1/2/NF-κB activation, cell growth, and migration. CONCLUSION: An increase in ILK non-canonically promotes ERK1/2/NF-κB activation and leads to the growth of gastric cancer cells.

Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-γ signaling.

Helicobacter pylori infection not only induces gastric inflammation but also increases the risk of gastric tumorigenesis. IFN-γ has antimicrobial effects; however, H. pylori infection elevates IFN-γ-mediated gastric inflammation and may suppress IFN-γ signaling as a strategy to avoid immune destruction through an as-yet-unknown mechanism. This study was aimed at investigating the mechanism of H. pylori-induced IFN-γ resistance. Postinfection of viable H. pylori decreased IFN-γ-activated signal transducers and activators of transcription 1 and IFN regulatory factor 1 not only in human gastric epithelial MKN45 and AZ-521 but also in human monocytic U937 cells. H. pylori caused an increase in the C-terminal tyrosine phosphorylation of Src homology-2 domain-containing phosphatase (SHP) 2. Pharmacologically and genetically inhibiting SHP2 reversed H. pylori-induced IFN-γ resistance. In contrast to a clinically isolated H. pylori strain HP238, the cytotoxin-associated gene A (CagA) isogenic mutant strain HP238(CagAm) failed to induce IFN-γ resistance, indicating that CagA regulates this effect. Notably, HP238 and HP238(CagAm) differently caused SHP2 phosphorylation; however, imaging and biochemical analyses demonstrated CagA-mediated membrane-associated binding with phosphorylated SHP2. CagA-independent generation of reactive oxygen species (ROS) contributed to H. pylori-induced SHP2 phosphorylation; however, ROS/SHP2 mediated IFN-γ resistance in a CagA-regulated manner. This finding not only provides an alternative mechanism for how CagA and ROS coregulate SHP2 activation but may also explain their roles in H. pylori-induced IFN-γ resistance.

Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production.

Autophagy facilitates cytokine-induced ICAM-1 expression.

ICAM-1 can be induced by inflammatory cytokines such as IFN-γ and TNF-α. This study investigated whether autophagy regulates ICAM-1 given that autophagy facilitates signaling of these two cytokines. Exogenous IFN-γ induced ICAM-1 in human lung epithelial A549 cells carrying wild type p53, a transcription factor reported for ICAM-1, but not in PC14PE6/AS2 (AS2) cells carrying mutated p53. However, IFN-γ also induced ICAM-1 in A549 cells with short hairpin RNA-silenced p53. No changes in IFN-γ receptor expression were observed in AS2 cells, but IFN-γ-activated Jak2/STAT1/IFN regulatory factor 1 was markedly decreased. In AS2 cells, increased levels of reactive oxygen species induced the activation of Src homology domain-containing phosphatase 2 (SHP2), while SHP2 was essential for IFN-γ resistance. AS2 cells showed autophagy resistance, and the manipulation of the autophagy pathway altered IFN-γ resistance. Aberrant Bcl-2 expression and mammalian target of rapamycin activation contributed to both autophagy resistance and IFN-γ resistance. Autophagy, but not p53, also modulated TNF-α-induced NF-κB activation and ICAM-1 expression. Inhibiting autophagy decreased the adhesion of human monocytic U937 cells to IFN-γ-treated A549 cells. These results demonstrated that IFN-γ and TNF-α induced ICAM-1 expression through a common pathway that was regulated by autophagy, but not p53.

Uropathogenic Escherichia coli causes cortical tubular necrotic cell death and the release of macrophage migration inhibitory factor.

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is deregulated in acute kidney injury (AKI) through an unknown mechanism. In the present study, we used a previously described mouse model of ascending urinary tract infection in which uropathogenic Escherichia coli (UPEC) were transurethrally inoculated to induce kidney infections. Here, we show that urinary MIF was upregulated during AKI while MIF was abundantly expressed in the renal cortical tubules and that UPEC infection caused a decrease in tubular MIF. Infections with UPEC in vitro caused MIF release in a cell type-dependent manner, which was independent of receptor-mediated internalization, signal transduction, and transcription. Indeed, UPEC infection-induced necrotic cell death in vitro and in vivo correlated with extracellular acidification and processed MIF secretion. These data suggest that MIF is released by necrotic renal cortical tubular cells during UPEC infection.

Glycogen synthase kinase-3ß inactivation is an intracellular marker and regulator for endotoxemic neutrophilia.

Neutrophilia, defined as a large number of neutrophils in the circulating blood, is caused by increased differentiation and survival from activation-induced apoptosis. Regulation of apoptosis is essential for neutrophil homeostasis; however, the molecular signaling that regulates this process needs further investigation. Unlike TLR4 wild-type C3H/HeN mice, TLR4 mutated C3H/HeJ mice were insusceptible to LPS-induced blood neutrophilia. LPS prevented constitutive apoptosis in neutrophils and partly involved a blockade of the mitochondrial pathway including mitochondria transmembrane potential loss, myeloid cell leukemia sequence (Mcl) 1 degradation, and caspase-3 activation. In apoptotic neutrophils, glycogen synthase kinase (GSK)-3ß was activated, and inhibiting GSK-3ß decreased Mcl-1 degradation and apoptosis. LPS caused p38 MAPK-, JNK-, and PI3K/AKT-mediated Mcl-1 stabilization and prevented apoptosis, and LPS induced GSK-3ß inactivation mainly through p38 MAPK and PI3K/AKT. Neutrophils in the neutrophilia showed increased GSK-3ß inactivation and Mcl-1 stabilization accompanied by activation of p38 MAPK, JNK, and AKT. Notably, LPS-induced ROS generation can partly facilitate p38 MAPK/JNK/AKT activation to regulate GSK-3ß-mediated Mcl-1 stability, apoptosis, and neutrophilia. These results demonstrate that the molecular basis of endotoxemic neutrophilia is through a direct action on neutrophils involving GSK-3ß inactivation to prevent constitutive apoptosis.

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses.

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H(2)O(2))-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), a chloromethyl derivative of H(2)DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H(2)O(2)-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H(2)O(2)-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.

Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation.

Aberrant levels of reactive oxygen species (ROS) rapidly generated from NADPH oxidase (NOX) activation can be cytotoxic due to activating pro-apoptotic signals. However, ROS also induce pro-survival autophagy through the engulfment of damaged mitochondria. This study is aimed at investigating the cytoprotective role of albumin against NOX/ROS-induced autophagy and apoptosis under serum starvation. Serum starvation induced apoptosis following a myeloid cell leukemia sequence 1 (Mcl-1)/Bax imbalance, loss of the mitochondrial transmembrane potential, and caspase activation accompanied by pro-survival autophagy following canonical inhibition of mammalian target of rapamycin complex 1 (mTORC1). Aberrant ROS generation, initially occurring through NOX, facilitated mitochondrial damage, autophagy, and apoptosis. Autophagy additionally regulated the accumulation of ROS-generating mitochondria. NOX/ROS permitted p38 mitogen-activated protein kinase (p38 MAPK)-regulated mitochondrial apoptosis, accompanied by non-canonical induction of autophagy. In addition, activation of glycogen synthase kinase (GSK)-3ß by NOX/ROS-inactivated Akt facilitated a decrease in Mcl-1, followed by mitochondrial apoptosis as well as autophagy. Restoring albumin conferred an anti-oxidative effect against serum starvation-deregulated NOX, p38 MAPK, and Akt/GSK-3ß/Mcl-1/caspase-3 signaling. Albumin also prevented autophagy by sustaining mTORC1. These results indicate an anti-oxidative role for albumin via preventing NOX/ROS-mediated mitochondrial signaling to stimulate apoptosis as well as autophagy. Autophagy, initially induced by canonical inhibition of mTORC1 and enhanced by non-canonical mitochondrial damage, acts physically as a pro-survival mechanism.

Inhibiting glycogen synthase kinase-3 decreases 12-O-tetradecanoylphorbol-13-acetate-induced interferon-γ-mediated skin inflammation.

Glycogen synthase kinase-3 (GSK-3) facilitates interferon (IFN)-γ signaling. Because IFN-γ is involved in inflammatory skin diseases, such as psoriasis, the aim of this study was to investigate the pathogenic role of GSK-3 in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IFN-γ-mediated ear skin inflammation. TPA (3 µg per ear) induced acute skin inflammation in the ears of C57BL/6 mice, including edema, infiltration of granulocytes but not T cells, and IFN-γ receptor 1-mediated deregulation of intercellular adhesion molecule 1 (CD54). TPA/IFN-γ induced GSK-3 activation, which in turn activated signal transducer and activator of transcription 1. Inhibiting GSK-3 pharmacologically, by administering 6-bromoindirubin-3'-oxime (1.5 µg per ear), and genetically, with lentiviral-based short-hairpin RNA, reduced TPA-induced acute skin inflammation but not T-cell infiltration. It is noteworthy that inhibiting GSK-3 decreased TPA-induced IFN-γ production and the nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, in CD3-positive T cells. In chronic TPA-induced skin inflammation, inhibiting GSK-3 attenuated epidermis hyperproliferation and dermis angiogenesis. These results demonstrate the dual role of GSK-3 in TPA-induced skin inflammation that is not only to facilitate IFN-γ signaling but also to regulate IFN-γ production. Inhibiting GSK-3 may be a potential treatment strategy for preventing such effects.

Regulation of SHP2 by PTEN/AKT/GSK-3ß signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer.

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3ß by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3ß/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3ß/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.

Vinca alkaloids cause aberrant ROS-mediated JNK activation, Mcl-1 downregulation, DNA damage, mitochondrial dysfunction, and apoptosis in lung adenocarcinoma cells.

Vinca alkaloids are clinically used to inhibit the growth of malignancy by interfering with microtubule polymerization. The purpose of this study was to identify the molecular mechanisms underlying growth inhibition as well as apoptosis in vinca alkaloid-treated lung adenocarcinoma cells. Consistent with nocodazole, treatment with vinorelbine (VNR) caused mitotic prometaphase arrest in a time-dependent manner, accompanied by cell apoptosis, dependent on both dose and time. VNR sequentially induced mitochondrial transmembrane potential (MTP) loss and caspase-dependent apoptosis following myeloid cell leukemia (Mcl) 1 downregulation. Prolonged activation of c-Jun N-terminal kinase (JNK) was required for vinca alkaloid- and nocodazole-induced apoptosis but not cell cycle arrest. Vinca alkaloids and nocodazole caused glutathione/reactive oxygen species (ROS) imbalance, and inhibiting ROS prevented prolonged JNK activation, decreased Mcl-1 levels, MTP loss, and apoptosis. Notably, cell size and granularity were enlarged in stimulated cells; unexpectedly, many ROS-producing mitochondria were accumulated followed by aberrant JNK-mediated mitochondrial dysfunction. Unlike cisplatin, which causes DNA damage in each phase of the cell cycle, VNR and nocodazole induced aberrant JNK-regulated DNA damage in prometaphase; however, inhibiting ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3-related) did not reverse mitotic arrest or apoptosis. These results demonstrate an essential role of ROS in vinca alkaloid-induced aberrant JNK-mediated Mcl-1 downregulation and DNA damage followed by mitochondrial dysfunction-related apoptosis but not mitotic arrest.

Glycogen synthase kinase-3ß is critical for interferon-α-induced serotonin uptake in human Jurkat T cells.

Dysregulation of glycogen synthase kinase (GSK)-3ß contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3ß regulation on IFN-α-induced 5-HTT functions. GSK-3ß short hairpin RNAs (shRNAs) or GSK-3ß inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3ß (Tyr216), which regulated 5-HT uptake. GSK-3ß knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3ß cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3ß activation and GSK-3ß-regulated 5-HT uptake. GSK-3ß signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3ß cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

Glycogen synthase kinase-3 facilitates con a-induced IFN-γ-- mediated immune hepatic injury.

Immune hepatic injury induced by Con A results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.