The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress

ABSTRACT Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1α markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1α or XBP1, disruption of PERK activity, or inhibition of eIF2α phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.

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Oxidative Damage to the Endoplasmic Reticulum is Implicated in Ischemic Neuronal Cell Death

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000089600.87125.ad ◽

2003 ◽

Vol 23 (10) ◽

pp. 1117-1128 ◽

Cited By ~ 88

Author(s):

Takeshi Hayashi ◽

Atsushi Saito ◽

Shuzo Okuno ◽

Michel Ferrand-Drake ◽

Robert L Dodd ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Protein Modification ◽

Neuronal Degeneration ◽

Neuronal Damage ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Transgenic Animals ◽

Initiation Factor

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2α (eIF2α) and RNA-dependent protein kinase-like ER eIF2α kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

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Human Cytomegalovirus Protein pUL38 Induces ATF4 Expression, Inhibits Persistent JNK Phosphorylation, and Suppresses Endoplasmic Reticulum Stress-Induced Cell Death

Journal of Virology ◽

10.1128/jvi.02307-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3463-3474 ◽

Cited By ~ 90

Author(s):

Baoqin Xuan ◽

Zhikang Qian ◽

Emi Torigoi ◽

Dong Yu

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Human Cytomegalovirus ◽

Initiation Factor ◽

Α Subunit ◽

Unfolded Protein ◽

Initiation Factor 2 ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT The endoplasmic reticulum (ER) is a key organelle involved in sensing and responding to stressful conditions, including those resulting from infection of viruses, such as human cytomegalovirus (HCMV). Three signaling pathways collectively termed the unfolded protein response (UPR) are activated to resolve ER stress, but they will also lead to cell death if the stress cannot be alleviated. HCMV is able to modulate the UPR to promote its infection. The specific viral factors involved in such HCMV-mediated modulation, however, were unknown. We previously showed that HCMV protein pUL38 was required to maintain the viability of infected cells, and it blocked cell death induced by thapsigargin. Here, we report that pUL38 is an HCMV-encoded regulator to modulate the UPR. In infection, pUL38 allowed HCMV to upregulate phosphorylation of PKR-like ER kinase (PERK) and the α subunit of eukaryotic initiation factor 2 (eIF-2α), as well as induce robust accumulation of activating transcriptional factor 4 (ATF4), key components of the PERK pathway. pUL38 also allowed the virus to suppress persistent phosphorylation of c-Jun N-terminal kinase (JNK), which was induced by the inositol-requiring enzyme 1 pathway. In isolation, pUL38 overexpression elevated eIF-2α phosphorylation, induced ATF4 accumulation, limited JNK phosphorylation, and suppressed cell death induced by both thapsigargin and tunicamycin, two drugs that induce ER stress by different mechanisms. Importantly, ATF4 overexpression and JNK inhibition significantly reduced cell death in pUL38-deficient virus infection. Thus, pUL38 targets ATF4 expression and JNK activation, and this activity appears to be critical for protecting cells from ER stress induced by HCMV infection.

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Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin

AJP Cell Physiology ◽

10.1152/ajpcell.00388.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C353-C361 ◽

Cited By ~ 39

Author(s):

Katsura Takano ◽

Yoshiyuki Tabata ◽

Yasuko Kitao ◽

Rika Murakami ◽

Hiroto Suzuki ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Cultured Cells ◽

Initiation Factor ◽

Substantia Nigra Pars Compacta ◽

Intracellular Camp ◽

Related Factor ◽

Renal Tubular Epithelium ◽

Enhanced Expression

Enhanced endoplasmic reticulum (ER) stress leads to cell death in various pathophysiological situations. During a search for compounds that regulate ER stress, we identified methoxyflavones, a group of flavonoids, as strong protective agents against ER stress. Analysis in mouse insulinoma MIN6 cells revealed that methoxyflavones mildly activated the eukaryotic initiation factor 2α and nuclear factor erythroid 2-related factor pathways, but not the XBP1 pathway, and induced downstream genes, including glucose-regulated protein (GRP) 78, a molecular chaperone in the ER. The protective effect of methoxyflavones was enhanced by agents that increase intracellular cAMP levels such as forskolin, dibutyryl-cAMP and IBMX, but suppressed by the protein kinase A (PKA) inhibitor H-89, suggesting involvement of the PKA pathway in the regulation of ER stress by methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal tubular epithelium and prevented tunicamycin-induced cell death. Furthermore, preadministration of tangeretin in mice enhanced expression of GRP78 in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and ER stress. These results suggest that methoxyflavones play an important role in the regulation of ER stress and could be a therapeutic target for the ER stress-related diseases.

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Induction of GRP78 by Ischemic Preconditioning Reduces Endoplasmic Reticulum Stress and Prevents Delayed Neuronal Cell Death

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000077641.41248.ea ◽

2003 ◽

Vol 23 (8) ◽

pp. 949-961 ◽

Cited By ~ 102

Author(s):

Takeshi Hayashi ◽

Atsushi Saito ◽

Shuzo Okuno ◽

Michel Ferrand-Drake ◽

Pak H Chan

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Ischemic Preconditioning ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Ischemic Tolerance ◽

Initiation Factor ◽

Tolerance Time ◽

Delayed Neuronal Cell Death

Although the endoplasmic reticulum (ER) is implicated in neuronal degeneration in some situations, its role in delayed neuronal cell death (DND) after ischemia remains uncertain. The authors speculated that ER stress is involved in DND, that it is reduced by ischemic preconditioning, and that ER stress reduction by preconditioning is due to ER molecular chaperone induction. The phosphorylation status of eukaryotic initiation factor 2α (eIF2α) and RNA-dependent protein kinase–like ER eIF2α kinase (PERK) was investigated in the rat hippocampus after ischemia with and without preconditioning. PERK is phosphorylated by ER stress, which phosphorylates eIF2α. To investigate the role of ER molecular chaperones in preconditioning, the authors examined GRP78 and GRP94 expression, both of which are ER chaperones that inhibit PERK phosphorylation, and compared their induction and ischemic tolerance time windows. Phosphorylation of eIF2α and PERK was confirmed after severe ischemia but was inhibited by preconditioning. After preconditioning, GRP78 was increased in the brain with a peak at 2 days, which corresponded with the ischemic tolerance time window. Immunoprecipitation and double staining demonstrated involvement of GRP78 in prevention of PERK phosphorylation. These results suggest that GRP78 induced by preconditioning may reduce ER stress and eventual DND after ischemia.

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IL-24 Promotes Apoptosis through cAMP-Dependent PKA Pathways in Human Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms19113561 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3561 ◽

Cited By ~ 7

Author(s):

Leah Persaud ◽

Jason Mighty ◽

Xuelin Zhong ◽

Ashleigh Francis ◽

Marifer Mendez ◽

...

Keyword(s):

Breast Cancer ◽

Cell Death ◽

Protein Kinase ◽

Er Stress ◽

Cancer Cell ◽

Mitogen Activated Protein Kinase ◽

Initiation Factor ◽

Breast Cancer Cell Lines ◽

Extrinsic Pathway ◽

Eif2α Phosphorylation

Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24.

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Inhibition of Autophagy Potentiated Hippocampal Cell Death Induced by Endoplasmic Reticulum Stress and its Activation by Trehalose Failed to be Neuroprotective

Current Neurovascular Research ◽

10.2174/1567202616666190131155834 ◽

2019 ◽

Vol 16 (1) ◽

pp. 3-11

Author(s):

Luisa Halbe ◽

Abdelhaq Rami

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Cell Damage ◽

Protective Mechanism ◽

Unfolded Proteins ◽

Neuronal Viability ◽

Hippocampal Cell ◽

Trigger Cell Death ◽

Autophagy Inhibition

Introduction: Endoplasmic reticulum (ER) stress induced the mobilization of two protein breakdown routes, the proteasomal- and autophagy-associated degradation. During ERassociated degradation, unfolded ER proteins are translocated to the cytosol where they are cleaved by the proteasome. When the accumulation of misfolded or unfolded proteins excels the ER capacity, autophagy can be activated in order to undertake the degradative machinery and to attenuate the ER stress. Autophagy is a mechanism by which macromolecules and defective organelles are included in autophagosomes and delivered to lysosomes for degradation and recycling of bioenergetics substrate. Materials and Methods: Autophagy upon ER stress serves initially as a protective mechanism, however when the stress is more pronounced the autophagic response will trigger cell death. Because autophagy could function as a double edged sword in cell viability, we examined the effects autophagy modulation on ER stress-induced cell death in HT22 murine hippocampal neuronal cells. We investigated the effects of both autophagy-inhibition by 3-methyladenine (3-MA) and autophagy-activation by trehalose on ER-stress induced damage in hippocampal HT22 neurons. We evaluated the expression of ER stress- and autophagy-sensors as well as the neuronal viability. Results and Conclusion: Based on our findings, we conclude that under ER-stress conditions, inhibition of autophagy exacerbates cell damage and induction of autophagy by trehalose failed to be neuroprotective.

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Polyhexamethylene Guanidine Phosphate Induces Apoptosis through Endoplasmic Reticulum Stress in Lung Epithelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031215 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1215

Author(s):

Mi Ho Jeong ◽

Mi Seon Jeon ◽

Ga Eun Kim ◽

Ha Ryong Kim

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Epithelial Cell ◽

Er Stress ◽

Lung Fibrosis ◽

A549 Cells ◽

Polyhexamethylene Guanidine ◽

Lung Epithelial ◽

Epithelial Cell Death ◽

Guanidine Phosphate

Airway epithelial cell death contributes to the pathogenesis of lung fibrosis. Polyhexamethylene guanidine phosphate (PHMG-p), commonly used as a disinfectant, has been shown to be strongly associated with lung fibrosis in epidemiological and toxicological studies. However, the molecular mechanism underlying PHMG-p-induced epithelial cell death is currently unclear. We synthesized a PHMG-p–fluorescein isothiocyanate (FITC) conjugate and assessed its uptake into lung epithelial A549 cells. To examine intracellular localization, the cells were treated with PHMG-p–FITC; then, the cytoplasmic organelles were counterstained and observed with confocal microscopy. Additionally, the organelle-specific cell death pathway was investigated in cells treated with PHMG-p. PHMG-p–FITC co-localized with the endoplasmic reticulum (ER), and PHMG-p induced ER stress in A549 cells and mice. The ER stress inhibitor tauroursodeoxycholic acid (TUDCA) was used as a pre-treatment to verify the role of ER stress in PHMG-p-induced cytotoxicity. The cells treated with PHMG-p showed apoptosis, which was inhibited by TUDCA. Our results indicate that PHMG-p is rapidly located in the ER and causes ER-stress-mediated apoptosis, which is an initial step in PHMG-p-induced lung fibrosis.

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c-Jun Inhibits Thapsigargin-Induced ER Stress Through Up-Regulation of DSCR1/Adapt78

Experimental Biology and Medicine ◽

10.3181/0803-rm-84 ◽

2008 ◽

Vol 233 (10) ◽

pp. 1289-1300 ◽

Cited By ~ 25

Author(s):

Peng Zhao ◽

Xiaoyan Xiao ◽

Agnes S. Kim ◽

M. Fatima Leite ◽

Jinxia Xu ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Wild Type Mouse ◽

Mouse Fibroblast ◽

Expression Levels ◽

Mouse Fibroblasts ◽

The Unfolded Protein Response ◽

Calcineurin Activity

The endoplasmic reticulum (ER) is exquisitely sensitive to changes in its internal environment. Various conditions, collectively termed “ER stress”, can perturb ER function, leading to the activation of a complex response known as the unfolded protein response (UPR). Although c-Jun N-terminal kinase (JNK) activation is nearly always associated with cell death by various stimuli, the functional role of JNK in ER stress-induced cell death remains unclear. JNK regulates gene expression through the phosphorylation and activation of transcription factors, such as c-Jun. Here, we investigated the role of c-Jun in the regulation of ER stress-related genes. c-Jun expression levels determined the response of mouse fibroblasts to ER stress induced by thapsigargin (TG, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase). c-jun−/− mouse fibroblast cells were more sensitive to TG-induced cell death compared to wild-type mouse fibroblasts, while reconstitution of c-Jun expression in c-jun−/− cells (c-Jun Re) enhanced resistance to TG-induced cell death. The expression levels of ER chaperones Grp78 and Gadd153 induced by TG were lower in c-Jun Re than in c-jun−/− cells. Moreover, TG treatment significantly increased calcineurin activity in c-jun−/− cells, but not in c-Jun Re cells. In c-Jun Re cells, TG induced the expression of Adapt78, also known as the Down syndrome critical region 1 (DSCR1), which is known to block calcineurin activity. Taken together, our findings suggest that c-Jun, a transcription factor downstream of the JNK signaling pathway, up-regulates Adapt78 expression in response to TG-induced ER stress and contributes to protection against TG-induced cell death.

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Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator

Endocrinology ◽

10.1210/en.2012-1418 ◽

2012 ◽

Vol 153 (11) ◽

pp. 5275-5284 ◽

Cited By ~ 41

Author(s):

Jun Lu ◽

Qinghua Wang ◽

Lianghu Huang ◽

Huiyue Dong ◽

Lingjing Lin ◽

...

Keyword(s):

Stem Cells ◽

Endoplasmic Reticulum ◽

Mesenchymal Stem Cells ◽

Protein Kinase ◽

Er Stress ◽

Human Mesenchymal Stem Cells ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Human Umbilical Cord ◽

Amp Activated Protein Kinase

Abstract Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

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