CAAT/enhancer Binding Protein Homologous Protein Research Articles

Editor's Note: Role of CAAT/Enhancer Binding Protein Homologous Protein in Panobinostat-mediated Potentiation of Bortezomib-induced Lethal Endoplasmic Reticulum Stress in Mantle Cell Lymphoma Cells.

Purpose: Bortezomib induces unfolded protein response (UPR) and endoplasmic reticulum stress, as well as exhibits clinical activity in patients with relapsed and refractory mantle cell lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of the pan-histone deacetylase inhibitor panobinostat and bortezomib against human, cultured, and primary MCL cells. Experimental Design: Immunoblot analyses, reverse transcription-PCR, and immunofluorescent and electron microscopy were used to determine the effects of panobinostat on bortezomib-induced aggresome formation and endoplasmic reticulum stress in MCL cells. Results: Treatment with panobinostat induced heat shock protein 90 acetylation; depleted the levels of heat shock protein 90 client proteins, cyclin-dependent kinase 4, c-RAF, and AKT; and abrogated bortezomib-induced aggresome formation in MCL cells. Panobinostat also induced lethal UPR, associated with induction of CAAT/enhancer binding protein homologous protein (CHOP). Conversely, knockdown of CHOP attenuated panobinostat-induced cell death of MCL cells. Compared with each agent alone, cotreatment with panobinostat increased bortezomib-induced expression of CHOP and NOXA, as well as increased bortezomib-induced UPR and apoptosis of cultured and primary MCL cells. Cotreatment with panobinostat also increased bortezomib-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft. Conclusion: These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib. Clin Cancer Res; 16(19); 4742–54. ©2010 AACR.

Knockdown of KIAA1199 attenuates growth and metastasis of hepatocellular carcinoma

Accumulating evidence indicates that KIAA1199 plays a vital role in tumor progression. However, the role of KIAA1199 in hepatocellular carcinoma (HCC) still remains unknown. In this study, we found that KIAA1199 was upregulated in human HCC tissues and in highly metastatic HCC cell lines. Furthermore, the expression of KIAA1199 was significantly correlated with tumor size and metastasis in HCC. Knockdown of KIAA1199 inhibited cell proliferation and migration in vitro, and suppressed tumorigenicity and lung metastasis in vivo. In addition, silencing of KIAA1199 induced G1 phase arrest by reducing cyclinD1 expression. Moreover, KIAA1199 knockdown induced apoptosis by activating endoplasmic reticulum (ER) stress, which was based on the upregulation of ER stress markers, activating transcription factor 4 (ATF4) and CAAT/enhancer-binding protein homologous protein (CHOP). In conclusion, our data demonstrated that KIAA1199 knockdown inhibited the growth and metastasis of HCC.

CAAT/enhancer binding protein-homologous protein deficiency attenuates liver ischemia/reperfusion injury in mice.

Ischemia/reperfusion injury (IRI) is one of the main causes of liver dysfunction after liver surgery. Involvement of endoplasmic reticulum (ER) stress in various diseases has been demonstrated, and CAAT/enhancer binding protein-homologous protein (CHOP) is a transcriptional regulator that is induced by ER stress. It is also a key regulator of ER stress-mediated apoptosis. The aim of this study was to investigate the role of CHOP in liver IRI. Wild type (WT) and CAAT/enhancer binding protein-homologous protein knockout (CHOP-/-) mice were subjected to 70% liver warm ischemia/reperfusion for 60 minutes. At different times after reperfusion, liver tissues and blood samples were collected for evaluation. Induction of ER stress including CHOP expression was ascertained. Liver damage was evaluated based on serum liver enzymes, liver histology, and neutrophil infiltration. Hepatocyte death including apoptosis was assessed. Liver warm IRI induced ER stress in both WT and CHOP-/- mice. In addition, CHOP expression was up-regulated in WT mice. At 6 hours after reperfusion, liver damage was attenuated in CHOP-/- mice. On the basis of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, apoptotic and necrotic cells were significantly reduced in CHOP-/- mice. CHOP deficiency also reduced the cleavage of caspase 3 and expression of the proapoptotic protein B cell lymphoma 2-associated X protein. Liver IRI induces CHOP expression, and CHOP deficiency attenuates liver IRI by inhibiting apoptosis. Elucidation of the function of CHOP in liver IRI may contribute to further investigation for a therapy against liver IRI associated with the ER stress pathway. Liver Transplantation 24 645-654 2018 AASLD.

Mechanism of Astragalus polysaccharides in attenuating insulin resistance in Rats with type 2 diabetes mellitus via the regulation of liver microRNA‑203a‑3p.

Insulin resistance (IR) is a common feature of type 2 diabetes mellitus (T2DM). Astragalus polysaccharides (APS) is a natural medicine that is used to treat T2DM. However, the mechanism by which APS regulates micro (mi)RNA in the treatment of IR has not been investigated. The purpose of the present study was to investigate differential miRNA expression between normal, T2DM model and APS treatment rats, as well as changes in miRNA and its downstream gene expression levels after APS treatment in T2DM Goto Kakizaki (GK) rats. Results suggested that miRNA (miR)-203a-3p expression level was significantly decreased in the liver of T2DM GK rats. Furthermore, it was identified that glucose-regulated protein (GRP)78 was the target gene of miR-203a-3p. GRP78 mRNA and protein expression levels of GRP78, CAAT-enhancer-binding protein homologous protein (CHOP), phosphorylated-c-Jun N-terminal kinase (pJNK)1, and caspase-12 were significantly increased in the liver of T2DM GK rats. Furthermore, miR-203a-3p was upregulated following APS treatment, and the protein expression levels of GRP78, CHOP, pJNK1 and caspase-12 were significantly decreased. In addition, miR-203a-3p overexpression in IR cells decreased the protein expression levels of these factors and anti-miR-203a-3p produced the opposite result. These findings provided evidence that miR-203a-3p may have a functional role in endoplasmic reticulum stress (ERS) signaling in the liver of T2DM GK rats. In addition, APS attenuated IR in T2DM, likely through upregulating or maintaining the miR-203a-3p expression levels, decreasing GRP78 mRNA and protein expression levels and regulating the protein expression of the ERS signaling pathway.

Discovery of (4-bromophenyl)(3-hydroxy-4-methoxyphenyl)methanone through upregulating hTERT induces cell apoptosis and ERS

Dominant-negative mutants of telomerase hTERT were demonstrated to have selective effects in tumor cells. However, no any effective and highly selective hTERT inhibitor has been developed so far. We focused on developing new hTERT modulators and synthesized a small molecular compound, named (4-bromophenyl)(3-hydroxy-4-methoxyphenyl)methanone. Our in vitro studies found that title compound showed high inhibitory activity against telomerase, had high antiproliferative capacity on SMMC-7721 cells with IC50 value 88 nm, and had no obvious toxic effect on human normal hepatocyte cells with IC50 value 10 μM. Our in vivo studies showed that this compound significantly inhibited tumor growth in xenograft tumor models. The further molecular mechanisms of title compound inhibition SMMC-7721 cell proliferation by modulating hTERT were explored; the results showed that endoplasmic reticulum stress (ERS) through ER over response (EOR) activates the expression of hTERT, and then induces ERS, which is believed to be intricately associated with oxidative stress and mitochondrial dysfunction, resulting in apoptotic cell death, thereby modulating the expression of downstream signaling molecules including CHOP (CAAT/enhancer-binding protein homologous protein)) and mitochondrion pathway of apoptosis, leading to inhibition of cell proliferation.

α7β1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise.

The α7β1 integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously demonstrated that expression of the α7BX2 integrin subunit (MCK:α7BX2) preserves muscle integrity and enhances myofiber cross-sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the α7BX2-integrin contributes to improvements in muscle growth after exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild-type or transgenic mice under sedentary conditions and 3 h following an acute bout of downhill running. Genes with false discovery rate probability values below the cutoff of P < 0.05 (n = 73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in endoplasmic reticulum protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression alone or in combination with exercise (P < 0.05). A significant increase in HSPA5 protein and a decrease in CAAT-enhancer-binding protein homologous protein (CHOP) were detected in transgenic muscle (P < 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5 The results from this study suggest that the α7β1 integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise.

Mitomycin C induces apoptosis in human epidural scar fibroblasts after surgical decompression for spinal cord injury.

Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C (1, 5, 10, 20, 40 μg/mL) for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 (Z-IETD-FMK and Z-LEHD-FMK, respectively) did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein (CHOP) and caspase-4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.

Effect of dexmedetomidine on hippocampal neuronal apoptosis in endotoxemic rats

Objective To evaluate the effect of dexmedetomidine on hippocampal neuronal apoptosis in endotoxemic rats. Methods Twenty-four pathogen-free male Sprague-Dawley rats, aged 6 weeks, weighing 200-250 g, were randomly divided into 3 groups(n=8 each)using a random number table: control group(group C), endotoxemia group(group E), and dexmedetomidine group(group D). Dexmedetomidine 50 μg/kg was injected intraperitoneally, and lipopolysaccharide 5 mg/kg was injected via the caudal vein 30 min later in group D. Normal saline 2 ml was injected intraperitoneally, and lipopolysaccharide 5 mg/kg was injected via the caudal vein 30 min later in group E. Normal saline 2 ml was injected intraperitoneally, and normal saline 2 ml was injected via the caudal vein 30 min later in group C. At 12 h after the model was successfully established, Morris water maze test was used to assess the cognitive function.The escape latency, swimming distance and frequency of crossing the original platform were recorded, and the swimming speed was calculated.The rats were then sacrificed, and hippocampi were harvest for determination of neuronal apoptosis(by TUNEL)and expression of glucose-regulated protein 78(GRP78), CAAT/enhancer-binding protein homologous protein(CHOP)and caspase-12(by immunohistochemistry). The apoptosis index(AI)was calculated. Results There was no significant difference in the swimming speed between the 3 groups(P>0.05). Compared with group C, the escape latency and swimming distance were significantly prolonged, the frequency of crossing the original platform was significantly decreased, and the AI was significantly increased, and the expression of GRP78, CHOP, and caspase-12 was significantly up-regulated in E and D groups(P<0.05). Compared with group E, the escape latency and swimming distance were significantly shortened, the frequency of crossing the original platform was significantly increased, and the AI was significantly decreased, and the expression of GRP78, CHOP, and caspase-12 was significantly down-regulated in group D(P<0.05). Conclusion Dexmedetomidine reduces cognitive dysfunction probably through reducing hippocampal neuronal apoptosis mediated by endoplasmic reticulum stress in endotoxemic rats. Key words: Dexmedetomidine; Endotoxemia; Apoptosis; Hippocampus; Neurons

Hispolon from Phellinus linteus induces apoptosis and sensitizes human cancer cells to the tumor necrosis factor-related apoptosis-inducing ligand through upregulation of death receptors

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in non‑malignant cells. However, certain type of cancer cells are resistant to TRAIL‑induced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAIL‑induced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAIL‑induced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase‑3, caspase‑8 and caspase‑9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl‑2 and Bcl‑xL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a non‑cell type‑specific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of anti‑apoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.

Dihydroartemisinin-Induced Apoptosis is Associated with Inhibition of Sarco/Endoplasmic Reticulum Calcium ATPase Activity in Colorectal Cancer.

Dihydroartemisinin (DHA) is a promising anti-cancer compound capable of inhibiting proliferation and inducing apoptosis of various cancer cells, including colorectal cancer. However, the molecular mechanisms have not been well understood. This study aimed to explore the underlying mechanism of DHA-induced apoptosis in HCT-116 cells. Cell counting kit-8 assay and flow cytometry analysis confirmed that DHA inhibited proliferation, arrested cell cycle at G0/G1 phase, and enhanced apoptosis in HCT-116 cells. Fluo-3/AM-stained flow cytometry assay revealed that the intracellular Ca(2+) concentration of HCT-116 cells was increased significantly after DHA treatment. Meanwhile, the activity of sarco/endoplasmic reticulum calcium ATPase (SERCA) was appeared to be reduced in a dose-dependent manner. We further detected the upregulated expression of CAAT/enhancer binding protein homologous protein (CHOP) in DHA-treated HCT-116 cells. Conversely, silencing CHOP resulted in a decrease of DHA-induced apoptosis. In addition, the expression of Bax in cytoplasm was elevated significantly along with the sharply decline of Bcl-2 expression in DHA-treated HCT-116 cells. Moreover, the distributions of Bid on mitochondria were increased, accompanied by the activation of caspase-3 in the presence of DHA. Overall, our data indicated that DHA triggered endoplasmic reticulum (ER) stress through inhibiting SERCA activity to release intracellular Ca(2+) from ER, the upregulated expression of CHOP activated mitochondrial apoptosis pathway to induce apoptosis of HCT-116 cells. Therefore, our findings provide a theoretical foundation for DHA as a potential candidate in treatment of colorectal cancer.

Role of Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Contrast-Induced Renal Tubular Cell Apoptosis

Background: Renal tubular cell apoptosis is a key mechanism of contrast-induced acute kidney injury. It has been reported that endoplasmic reticulum (ER) stress is the underlying mechanism of high osmolar contrast-induced renal tubular cell apoptosis. Whether ER stress is involved in low osmolar contrast-induced renal tubular cell injury remains unclear. In the present study, the roles of ER stress in iopromide-induced (a low osmolar contrast) renal tubular cell apoptosis and the effects of N-acetylcysteine (NAC) on ER stress were investigated. Methods: NRK-52E cells were exposed to different concentrations of iopromide [50, 100 and 150 mg iodine (I)/ml] for 4 h. In a separate experiment, NRK-52E cells were exposed to iopromide (100 mg I/ml, 4 h) with or without NAC (10 mmol/l). NAC was added 1 h before incubation with iopromide. Apoptosis was determined by Hoechst staining and flow cytometry. The intracellular formation of reactive oxygen species (ROS) was detected by confocal microscopy with fluorescent probe CM-H2DCFDA. The expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) was determined by Western blot. Results: Iopromide induced NRK-52E cell apoptosis in a concentration-dependent manner. The intracellular ROS production increased significantly following iopromide exposure in the NRK-52E cells. Significantly increased expressions of GRP78 and CHOP were observed in the NRK-52E cells exposed to iopromide for 4 h; NAC attenuated iopromide-induced NRK-52E cell apoptosis by inhibiting the overproduction of intracellular ROS and subsequently suppressing the overexpression of GRP78 and CHOP. Conclusion: ROS-mediated ER stress is involved in contrast-induced renal tubular cell apoptosis.

Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity

The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stresses that include hypoxia, nutrient deprivation, and change in redox status. These stress stimuli are common in many tumors and thus targeting components of the UPR signaling is an attractive therapeutic approach. We have identified a first-in-class, small molecule inhibitor of the eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3) or PERK, one of the three mediators of UPR signaling. GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity with an IC(50) of 0.9 nmol/L. It is highly selective for PERK with IC(50) values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells with an IC(50) in the range of 10-30 nmol/L as shown by inhibition of stress-induced PERK autophosphorylation, eIF2α substrate phosphorylation, together with corresponding decreases in ATF4 and CAAT/enhancer binding protein homologous protein (CHOP) in multiple cell lines. Oral administration of GSK2656157 to mice shows a dose- and time-dependent pharmacodynamic response in pancreas as measured by PERK autophosphorylation. Twice daily dosing of GSK2656157 results in dose-dependent inhibition of multiple human tumor xenografts growth in mice. Altered amino acid metabolism, decreased blood vessel density, and vascular perfusion are potential mechanisms for the observed antitumor effect. However, despite its antitumor activity, given the on-target pharmacologic effects of PERK inhibition on pancreatic function, development of any PERK inhibitor in human subjects would need to be cautiously pursued in cancer patients.

Signaling Pathways Involved in Endoplasmic Reticulum Stress-Induced Neuronal Apoptosis

ABSTRACTIn eukaryotic cells, the endoplasmic reticulum (ER) is a very critical site for synthesis, folding, modification of protein, and calcium homeostasis. The ER responds to factors that perturb ER function such as the accumulation of unfolded proteins (ER stress) by activating unfolded protein response to relieve the stress. However, chronic or unresolved ER stress can induce neuronal apoptosis by activating c-Jun N-terminal kinase (JNK), glycogen synthase kinase 3/3β (GSK3/3β), CAAT/enhancer binding protein homologous protein (CHOP), and caspase-12 pathway. Research related to ER stress will provide therapeutic implications in neurological diseases.

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.

Abstract 2078: Treatment with auranofin induces oxidative and lethal endoplasmic reticulum (ER) stress exerting single agent activity against mantle cell lymphoma cells

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis. In the present studies, we determined for the first time the lethal activity of AF (100 to 1000 nM) and its underlying mechanism(s) in cultured and primary Mantle Cell Lymphoma (MCL) cells. We demonstrate that treatment with AF in JeKo-1 and Z138C cells induced 2 to 3.5 fold increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 35-40%) without alteration in the expression of TRR. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in heme oxygenase-1 (HMOX1) expression. Microarray analysis revealed that AF treatment significantly upregulated ER stress and oxidative stress inducible genes including that of the ER-stress induced pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein), ATF3 (Activated Transcription Factor 3) and Glutamate-cysteine ligase (GCL), modifier subunit (GCLM), the rate-limiting enzyme in the glutathione (GSH) biosynthesis. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (&amp;lt; 15% apoptosis) (p &amp;lt; 0.01). Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, HSF1 and p97, resulting in hyperacetylation of hsp90 and depletion of the levels of hsp90 client proteins including HDAC6, AKT and Cyclin D1 in MCL cells. Co-treatment of MCL cells with AF and the proteasome inhibitor carfilzomib (CZ) resulted in synergistic apoptosis in cultured MCL cells and significantly more apoptosis in primary MCL cells, than treatment with each agent alone. Based on the induction of GCLM and anti-oxidant response genes following AF treatment, we tested the anti-MCL activity of the combination of AF and the GCL inhibitor Buthionine sulphoximine (BSO), against MCL cells. Co-treatment with BSO and AF resulted in significantly more apoptosis of MCL cells than treatment with each agent alone. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with MCL as a single agent and in combination with other ER and oxidative stress-inducing agents, especially GCL inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2078. doi:1538-7445.AM2012-2078

Endoplasmic reticulum stress modulates nicotine‐induced extracellular matrix degradation in human periodontal ligament cells

Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB. These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.

Endoplasmic reticulum stress mediates aristolochic acid I-induced apoptosis in human renal proximal tubular epithelial cells

Aristolochic acid (AA), derived from the Aristolochia species, has been associated with aristolochic acid nephropathy (AAN), which has emerged as a worldwide disease. Aristolochic acid I (AAI) is the main ingredient of AA, and the underlying mechanisms for AAI-induced nephrotoxicity are still unclear. In this study, we investigated whether endoplasmic reticulum (ER) stress was involved in AAI-induced nephrotoxicity. The results showed that treatment of HK-2 cells (a human proximal tubular epithelial cell line) with AAI caused an increase in eukaryotic initiation factor-2α (eIF2α) phosphorylation, X-box binding protein 1 (XBP1) mRNA splicing and the expression of glucose-regulated protein (GRP) 78 and CAAT/enhancer-binding protein-homologous protein (CHOP). These events represent typical markers of the ER stress-related signaling pathway. Pretreatment with 4-phenylbutyrate (4-PBA) or salubrinal (Sal) significantly inhibited AAI-induced apoptosis, indicating the role of ER stress in AAI-induced apoptosis. In addition, AAI-induced cell death followed an increase of reactive oxygen species (ROS) formation in HK-2 cells. Pretreatment with N-acetyl cysteine (NAC) or glutathione (GSH) significantly inhibited AAI-induced ER stress proteins and cell death, suggesting that ROS mediate AAI-induced ER stress. Taken together, these results suggest that the ER stress response is involved in apoptosis induced by AAI in HK-2 cells, thus offering a new insight into the nephrotoxicity of AAI.

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells

Apoptosis contributes to tubular epithelial cell death and atrophy in aldosterone (Aldo)-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells. Intracellular ROS generation was evaluated by 2′,7′-dichlorofluorescin diacetate fluorescence. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively. Aldo promoted tubular epithelial cell apoptosis, increased intracellular ROS production and induced ER stress, as evidenced by increased expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) in a dose- and time-dependent manner. Additionally, siRNA knockdown of CHOP and antioxidant N-acetyl-l-cysteine (NAC) attenuated ER stress-mediated apoptosis. NAC also could inhibit Aldo-induced expression of GRP78 and CHOP. Altogether, these observations suggest that Aldo induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells.

The endoplasmic reticulum stress response is involved in apoptosis induced by aloe-emodin in HK-2 cells

Aloe-emodin (AE; 1,8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) is one of the primary active compounds in total rhubarb anthraquinones (TRAs), which induce nephrotoxicity in rats. However, it is still not known whether AE has a similar effect on human kidney cells. In this study, 3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that AE decreases the viability of HK-2 cells (a human proximal tubular epithelial cell line) in a dose- and time-dependent manner. AE induced G2/M arrest of cell cycle in HK-2 cells, which was detected with propidium iodide (PI) staining. This apoptosis was further investigated by Hoechst staining, transmission electron microscopy (TEM), DNA fragmentation, and Annexin V/PI staining. Apoptosis of the cells was associated with caspase 3 activation, which was detected by Western blot analysis and a caspase activity assay. In addition, changes in the endoplasmic reticulum (ER) ultrastructure as observed by TEM showed the effects of AE on ER. Treatment with AE also resulted in an increase in eukaryotic initiation factor-2α (eIF-2α) phosphorylation, X-box binding protein 1 (XBP1) mRNA splicing, c-Jun N-terminal kinase (JNK) phosphorylation, glucose-regulated protein (GRP) 78 and CAAT/enhancer-binding protein-homologous protein (CHOP) accumulation. These results suggest that AE induces ER stress in HK-2 cells, which is involved in AE-induced apoptosis. In conclusion, AE induces apoptosis in HK-2 cells, and the ER stress is involved in AE-induced apoptosis in vitro. The implications of the toxic effects of AE for clinical use are unclear and these findings should be taken into account in the risk assessment for human exposure.

Treatment with Auranofin Induces Oxidative and Lethal Endoplasmic Reticulum (ER) Stress Exerting Single Agent Activity Against Primary CLL Cells

Abstract 929Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis, based upon its anti-inflammatory and immunosuppressive effects. In the present studies, we determined for the first time the lethal activity of AF (250 to 1000 nM) and its underlying mechanism(s) in CD19+ primary CLL cells, including those with del(17p13.1). We demonstrate that treatment with AF induced 20 to 40% increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 45%) without alteration in the expression of TRR, as determined in the AF treated cell lysates of CLL cells. AF treatment also increased the levels of thioredoxin binding protein 2. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in hemeoxygenase-1 (HO-1) levels. Thus, AF treatment perturbed the redox status in CLL cells. Consistent with increased levels of intracellular ROS, AF treatment also induced the markers of ER stress response including the induction of GRP78 and a sustained increase in the levels of the pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein). This was associated with induction of the levels of the pro-death BH3 only domain protein BIM, which is a known mediator of lethal ER stress. Exposure to 1000 nM AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment induced apoptosis to a similar extent in primary CLL cells with del(17p13.1) as compared to other genetic abnormalities. Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF also increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, resulting in hyperacetylation of hsp90 and depletion of the levels of HDAC6 in CLL cells. AF mediated hyperacetylation of hsp90 and α-tubulin was associated with proteasomal degradation of several of the CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Co-treatment of CLL cells with AF and the proteasome inhibitor carfilzomib (20 nM) significantly restored the levels of ZAP70, c-Raf, AKT and HDAC6. Finally, relatively lower levels of AF (250 nM) combined with the pan-HDAC inhibitor panobinostat (20 nM) or carfilzomib (5 nM) induced more cell death of primary CLL cells than each agent alone. These findings indicate that treatment of primary CLL cells with AF results in oxidative and ER stress. AF treatment also promotes the depletion of pro-survival CLL-relevant hsp90 client proteins. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with CLL including those with del(17p13.1). Disclosures:Reyes:Millennium, Sanofi Aventis: Consultancy.