scholarly journals PKR and GCN2 stress kinases promote an ER stress-independent eIF2α phosphorylation responsible for calreticulin exposure in melanoma cells

2018 ◽  
pp. e1466765 ◽  
Author(s):  
Paola Giglio ◽  
Mara Gagliardi ◽  
Nicola Tumino ◽  
Fernanda Antunes ◽  
Soraya Smaili ◽  
...  
Keyword(s):  
Er Stress ◽  
Melanoma Cells ◽  
Stress Kinases ◽  
Eif2α Phosphorylation

scholarly journals Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 153
Author(s):  
Min Seung Lee ◽  
So Hyun Lim ◽  
Ah-Ran Yu ◽  
Chi Yeon Hwang ◽  
Insug Kang ◽  
...  
Keyword(s):  
Er Stress ◽  
Dose Reduction ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Proteasome Inhibitors ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Melanoma Cells ◽  
Eif2α Phosphorylation ◽  
Pharmacological Interactions

Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.

Pinocembrin induces ER stress mediated apoptosis and suppresses autophagy in melanoma cells

2018 ◽  
Vol 431 ◽  
pp. 31-42 ◽  
Author(s):  
Yufei Zheng ◽  
Kai Wang ◽  
Yuqi Wu ◽  
Yifan Chen ◽  
Xi Chen ◽  
...  
Keyword(s):  
Er Stress ◽  
Melanoma Cells

scholarly journals The integrated stress response promotes B7H6 expression

2019 ◽  
Vol 98 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Akram Obiedat ◽  
Yoav Charpak-Amikam ◽  
Julie Tai-Schmiedel ◽  
Einat Seidel ◽  
Mohamed Mahameed ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Protease Inhibitors ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors ◽  
List Type ◽  
Mrna Levels ◽  
Integrated Stress Response ◽  
Eif2α Phosphorylation ◽  
Car T

Abstract The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. Key messages B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

scholarly journals Polyphenol-rich extract induces apoptosis with immunogenic markers in melanoma cells through the ER stress-associated kinase PERK

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Karol Prieto ◽  
Yu Cao ◽  
Eslam Mohamed ◽  
Jimena Trillo-Tinoco ◽  
Rosa A. Sierra ◽  
...  
Keyword(s):  
Er Stress ◽  
Stress Responses ◽  
Calcium Release ◽  
Melanoma Cells ◽  
Melanoma Tumor ◽  
Cancer Cell Death ◽  
Extracellular Release ◽  
Induced Apoptosis ◽  
Stress Mediators

Abstract Polyphenols elicit antitumor activities, in part, through the induction of anti- or pro-oxidant effects in cancer cells which promote priming of protective anti-tumor immunity. We recently characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) that stimulates in vivo antitumor responses against breast and melanoma tumor models via the promotion of immunogenic cancer cell death (ICD). However, the primary mediators whereby P2Et promotes ICD remained unknown. Here, we sought to elucidate the role that severe endoplasmic reticulum (ER) stress plays in mediating P2Et-induced apoptosis and ICD in murine melanoma cells. Our findings demonstrate a substantial selective induction of specific ER-stress mediators in B16-F10 melanoma cells treated with P2Et. While knockout of the ER stress-associated PKR-like ER kinase (PERK) prevented induction of apoptosis and expression of ICD markers in P2Et-treated cells, deletion of X-box binding protein 1 (Xbp1) did not. P2Et-driven activation of PERK in melanoma cells was found to promote ER-calcium release, disrupt mitochondrial membrane potential, and trigger upregulation of ICD drivers, surface calreticulin expression, and extracellular release of ATP and HMGB1. Notably, calcium release inhibition, but not targeting of PERK-driven integrated stress responses, prevented P2Et-induced apoptosis. Collectively, these results underline the central role of PERK-directed calcium release in mediating the antitumor and immunogenic actions of P2Et in melanoma cells.

scholarly journals Endoplasmic Reticulum Stress Signaling Comprises a G9a Inhibitor Tolerance Pathway and PERK Inhibition Increases Anti-Leukemia Activity of G9a Inhibitor in Leukemia Cells and Leukemia Stem-like Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1360-1360
Author(s):  
Jieun Jang ◽  
Ju-In Eom ◽  
Hoi-kyung Jeung ◽  
So-Young Seol ◽  
Haerim Chung ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Apoptotic Cell Death ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Eif2α Phosphorylation ◽  
Kg1 Cells

Abstract Background: Histone methyltransferase (HMTase) G9a regulates the transcription of multiple genes by primarily catalyzing dimethylation of histone H3 lysine 9 (H3K9me2), as well as several non-histone lysine sites. Recently, pharmacological and genetic targeting of the G9a was shown to be efficient in slowing down acute myeloid leukemia (AML) cell proliferation in a mouse model and human AML cell lines thus making this HMTase potential target for epigenetic therapy of AML. Activation of adaptive mechanisms to drug plays a crucial role in drug resistance and relapse by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The tolerance mechanism to HMTase regulation in leukemia cell is unclear yet. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Recent previous studies showed that pro-survival ER stress is induced in cancer cells and contributes to development of drug resistance. Methods: We investigated the levels of apoptosis and ER stress by G9a inhibitor BIX-01294 in leukemia cell lines. U937, cytarabine-resistant U937 (U937/AR) and KG1 were used. U937/AR cell line was established in our laboratory by exposing parental U937 cells to stepwise increasing concentrations of cytarabine. Results: We initially examined the expression of G9a in leukemia cell lines and the primary AML cells obtained from a patient at the different time point. In U937/AR cells and primary AML cells obtained at relapse, G9a expression was increased compare to that in U937 cells and primary AML cells obtained at diagnosis, respectively. G9a expression was also increased in KG1 cells. In both of U937 and U937/AR, apoptotic cell death was induced by BIX-01294 in a dose-dependent manner. In contrast, apoptotic cell death was minimal in KG1 cells which are enriched in cells expressing a leukemia stem cell phenotype (CD34+CD38-). To address the activation of ER stress response by BIX-01294 in leukemia cells, we examined the effect of BIX-01294 treatment on PERK and eIF2α protein expression and phosphorylation levels. We found that treatment of U937, U937/AR, KG1 cells with 3μM of BIX-01294 for 24h caused an upregulation of phosphorylated PERK and eIF2α. The upregulation of PERK phosphorylation was associated with a decrease in PERK protein levels after treatment. To further address the role of the PERK-eIF2α phosphorylation in BIX-01294 sensitivity, we examined whether PERK inhibition using small interfering RNA (siRNA) or specific inhibitor could sensitize cells to BIX-01294-mediated death. The siRNA against PERK effectively inhibited BIX-01294-mediated phosphorylation of PERK and eIF2α in U937 and U937/AR cells. The addition of PERK siRNA led to a significant increase in the extent of BIX-01294-induced apoptotic cell death in U937 (P = 0.0003) and U937/AR (P < 0.0001) as compared with that of BIX-01294 treatment alone. PERK inhibitor GSK260641 significantly increased BIX-01294-induced apoptotic cell death in U937 (P < 0.0001) and U937/AR (P = 0.006) cells. To our surprise, addition of PERK siRNA or GSK260641 increased the sensitivity of KG1 cells to BIX-01294-mediated death in a dose-dependent manner (P = 0.0003 for siRNA, P = 0.0053 for GSK260641). Conclusion: These data demonstrated that PERK-eIF2α activation has a pro-survival function to G9a inhibitor in leukemia cells and mediates resistance of AML stem cells to G9a inhibitor treatment. The PERK-eIF2α phosphorylation arm may represent a suitable target for combating resistance to G9a inhibitor in AML. The mechanisms underlying the increased sensitivity of AML cells with PERK inhibition to G9a inhibitor are unclear at present and are needed to define in further studies. Disclosures No relevant conflicts of interest to declare.

scholarly journals PERK-Mediated eIF2α Phosphorylation Contributes to The Protection of Dopaminergic Neurons from Chronic Heat Stress in Drosophila

2020 ◽  
Vol 21 (3) ◽  
pp. 845 ◽  
Author(s):  
Rosalie Elvira ◽  
Sun Joo Cha ◽  
Gyeong-Mu Noh ◽  
Kiyoung Kim ◽  
Jaeseok Han
Keyword(s):  
Heat Stress ◽  
Er Stress ◽  
Physiological Stress ◽  
Neuronal Cell Death ◽  
Heat Exposure ◽  
Neuronal Cell ◽  
Critical Factor ◽  
Initiation Factor ◽  
Eif2α Phosphorylation

Environmental high-temperature heat exposure is linked to physiological stress such as disturbed protein homeostasis caused by endoplasmic reticulum (ER) stress. Abnormal proteostasis in neuronal cells is a common pathological factor of Parkinson’s disease (PD). Chronic heat stress is thought to induce neuronal cell death during the onset and progression of PD, but the exact role and mechanism of ER stress and the activation of the unfolded protein response (UPR) remains unclear. Here, we showed that chronic heat exposure induces ER stress mediated by the PKR-like eukaryotic initiation factor 2α kinase (PERK)/eIF2α phosphorylation signaling pathway in Drosophila neurons. Chronic heat-induced eIF2α phosphorylation was regulated by PERK activation and required for neuroprotection from chronic heat stress. Moreover, the attenuated protein synthesis by eIF2α phosphorylation was a critical factor for neuronal cell survival during chronic heat stress. We further showed that genetic downregulation of PERK, specifically in dopaminergic (DA) neurons, impaired motor activity and led to DA neuron loss. Therefore, our findings provide in vivo evidence demonstrating that chronic heat exposure may be a critical risk factor in the onset of PD, and eIF2α phosphorylation mediated by PERK may contribute to the protection of DA neurons against chronic heat stress in Drosophila.

scholarly journals IRE1α-Dependent Decay of CReP/Ppp1r15b mRNA Increases Eukaryotic Initiation Factor 2α Phosphorylation and Suppresses Protein Synthesis

2015 ◽  
Vol 35 (16) ◽  
pp. 2761-2770 ◽  
Author(s):  
Jae-Seon So ◽  
Sungyun Cho ◽  
Sang-Hyun Min ◽  
Scot R. Kimball ◽  
Ann-Hwee Lee
Keyword(s):  
Protein Synthesis ◽  
Er Stress ◽  
Translational Control ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
The Unfolded Protein Response

The unfolded protein response (UPR) regulates endoplasmic reticulum (ER) homeostasis and protects cells from ER stress. IRE1α is a central regulator of the UPR that activates the transcription factor XBP1s through an unconventional splicing mechanism using its endoribonuclease activity. IRE1α also cleaves certain mRNAs containing XBP1-like secondary structures to promote the degradation of these mRNAs, a process known as regulated IRE1α-dependent decay (RIDD). We show here that the mRNA of CReP/Ppp1r15b, a regulatory subunit of eukaryotic translation initiation factor 2α (eIF2α) phosphatase, is a RIDD substrate. eIF2α plays a central role in the integrated stress response by mediating the translational attenuation to decrease the stress level in the cell. CReP expression was markedly suppressed in XBP1-deficient mice livers due to hyperactivated IRE1α. Decreased CReP expression caused the induction of eIF2α phosphorylation and the attenuation of protein synthesis in XBP1-deficient livers. ER stress also suppressed CReP expression in an IRE1α-dependent manner, which increased eIF2α phosphorylation and consequently attenuated protein synthesis. Taken together, the results of our study reveal a novel function of IRE1α in the regulation of eIF2α phosphorylation and the translational control.

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model

Reproduction ◽  
2021 ◽  
Author(s):  
Veronika Tandl ◽  
Denise Hoch ◽  
Julia Bandres-Meriz ◽  
Sanela Nikodijevic ◽  
Gernot Desoye ◽  
...  
Keyword(s):  
Er Stress ◽  
Early Pregnancy ◽  
Physiological Role ◽  
Brefeldin A ◽  
First Trimester ◽  
Fold Change ◽  
Low Oxygen ◽  
Placental Development ◽  
Eif2α Phosphorylation ◽  
The Unfolded Protein Response

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24h, while eIF2α protein and CHOP expression were downregulated (–28%; P < 0.05 and –24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48h by low oxygen (–61%; P < 0.05), but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

scholarly journals Zearalenone Induces Endoplasmic Reticulum Stress and Modulates the Expression of Phase I/II Enzymes in Human Liver Cells

Toxins ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 2 ◽  
Author(s):  
Jee Eun Yoon ◽  
Kwang Yong Lee ◽  
Jin Sil Seok ◽  
Wei Nee Cheng ◽  
Hyuk Cheol Kwon ◽  
...  
Keyword(s):  
Phase I ◽  
Er Stress ◽  
Human Liver ◽  
Fusarium Species ◽  
Control Level ◽  
Liver Cells ◽  
Protein Levels ◽  
Human Liver Cells ◽  
Eif2α Phosphorylation ◽  
Human Livers

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species; however, its mechanisms of action in human livers have not been fully elucidated. Thus, we investigated the toxic mechanisms of ZEN in human liver cells. HepG2 cells were treated with ZEN (0–40 μg/mL) for up to 24 h. A significant decrease in cell viability was observed after treatment with 20 and 40 μg/mL of ZEN, including a significant increase in apoptosis and reactive oxygen species production. ZEN increased GRP78 and CHOP, and eIF2α phosphorylation, indicating ER stress; elevated transcription of the autophagy-associated genes, beclin1 and LC3, and translation of LC3; and increased phase I metabolism by increasing PXR and CYP3A4. The protein expression level of CYP3A4 was higher with ZEN treatment up to 20 μg/mL, but remained at the control level after treatment with 40 μg/mL ZEN. In phase II metabolism, Nrf2 activation and UGT1A expression were increased with ZEN treatment up to 20 μg/mL. Treating cells with an ER stress inhibitor alleviated ZEN-induced cell death and autophagy, and inhibited the expression of phase I/II enzymes. Overall, high ZEN concentrations can modulate the expression of phase I/II enzymes via ER stress and reduced protein levels in human liver cells.

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