Maintain Endoplasmic Reticulum Homeostasis Research Articles

Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and drug resistance, yet few vulnerabilities of this state have been identified. Using selective small molecules as cellular probes, we show that induction of EMT greatly sensitizes cells to agents that perturb endoplasmic reticulum (ER) function. This sensitivity to ER perturbations is caused by the synthesis and secretion of large quantities of extracellular matrix (ECM) proteins by EMT cells. Consistent with their increased secretory output, EMT cells display a branched ER morphology and constitutively activate the PERK-eIF2α axis of the unfolded protein response (UPR). Protein kinase RNA-like ER kinase (PERK) activation is also required for EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression correlates strongly with both ECM and PERK-eIF2α genes, but not with other branches of the UPR. Taken together, our findings identify a novel vulnerability of EMT cells, and demonstrate that the PERK branch of the UPR is required for their malignancy. EMT drives tumor metastasis and drug resistance, highlighting the need for therapies that target this malignant subpopulation. Our findings identify a previously unrecognized vulnerability of cancer cells that have undergone an EMT: sensitivity to ER stress. We also find that PERK-eIF2α signaling, which is required to maintain ER homeostasis, is also indispensable for EMT cells to invade and metastasize.

The Caenorhabditis elegans HP1 family protein HPL-2 maintains ER homeostasis through the UPR and hormesis

Cellular adaptation to environmental changes and stress relies on a wide range of regulatory mechanisms that are tightly controlled at several levels, including transcription. Chromatin structure and chromatin binding proteins are important factors contributing to the transcriptional response to stress. However, it remains largely unknown to what extent specific chromatin factors influence the response to distinct forms of stress in a developmental context. One of the best characterized stress response pathways is the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). Here, we show that Caenorhabditis elegans heterochromatin protein like-2 (HPL-2), the homolog of heterochromatin protein 1 (HP1), down-regulates the UPR in the intestine. Inactivation of HPL-2 results in an enhanced resistance to ER stress dependent on the X-box binding protein 1 (XBP-1)/inositol requiring enzyme 1 branch of the UPR and the closely related process of autophagy. Increased resistance to ER stress in animals lacking HPL-2 is associated with increased basal levels of XBP-1 activation and ER chaperone expression under physiological conditions, which may in turn activate an adaptive response known as ER hormesis. HPL-2 expression in intestinal cells is sufficient to rescue stress resistance, whereas expression in neuronal cells negatively influenced the ER stress response through a cell-nonautonomous mechanism. We further show that the retinoblastoma protein homolog LIN-35 and the LIN-13 zinc finger protein act in the same pathway as HPL-2 to limit the ER stress response. Altogether, our results point to multiple functions for HP1 in different cell types to maintain ER homeostasis.

Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells

The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins, such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER-stressed cells. Here, we report the establishment of a system for monitoring the ER redox state in mammalian cells. The new ER redox-sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes the ER redox state. The mammalian ER redox-sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration, and Wolfram syndrome.

Theire-1ER Stress-Response Pathway is Required for Normal Secretory- Protein Metabolism inC. elegans

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress by adjusting the capacity of the ER to the load of ER-associated tasks. The UPR is important for maintaining ER homeostasis under extreme ER stress. UPR genes are important under normal growth conditions as well, but what they are required for under these conditions is less clear. Using C. elegans, we show that the ire-1/xbp-1 arm of the UPR plays a crucial role in maintaining ER plasticity and function also in the absence of external ER stress. We find that during unstressed growth conditions, loss of ire-1 or xbp-1 compromises basic ER functions required for the metabolism of secreted proteins, including translation, folding and secretion. Notably, by compromising ER-associated degradation (ERAD) and phagocytosis, loss of ire-1 hinders the clearance of misfolded proteins from the ER as well as the clearance of proteins that were secreted into the pseudocoleom. Whereas the basal activity of the UPR is beneficial under normal conditions, it accelerates the pathology caused by toxic Aβ protein in a C. elegans model of Alzheimer's disease. Taken together, our findings indicate that UPR genes are critical for maintaining secretory protein metabolism under normal growth conditions.

Selective Activation of ATF6 and PERK Endoplasmic Reticulum Stress Signaling Pathways Prevent Mutant Rhodopsin Accumulation

Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding. To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins. ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell. This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation.

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1--a G protein-coupled catecholamine receptor expressed in neurons--suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.

The impact of the unfolded protein response on human disease

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.

The homocysteine-inducible endoplasmic reticulum (ER) stress protein Herp counteracts mutant α-synuclein-induced ER stress via the homeostatic regulation of ER-resident calcium release channel proteins

Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases. The mechanisms that lead to ER stress and whereby ER stress contributes to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies. Here we show that knockdown of Herp (Homocysteine-inducible ER stress protein), an ER stress-inducible protein with an ubiquitin-like (UBL) domain, aggravates ER stress-mediated cell death induced by mutant α-synuclein (αSyn) that causes an inherited form of Parkinson's disease (PD). Functionally, Herp plays a role in maintaining ER homeostasis by facilitating proteasome-mediated degradation of ER-resident Ca(2+) release channels. Deletion of the UBL domain or pharmacological inhibition of proteasomes abolishes the Herp-mediated stabilization of ER Ca(2+) homeostasis. Furthermore, knockdown or pharmacological inhibition of ER Ca(2+) release channels ameliorates ER stress, suggesting that impaired homeostatic regulation of Ca(2+) channels promotes a protracted ER stress with the consequent activation of ER stress-associated apoptotic pathways. Interestingly, sustained upregulation of ER stress markers and aberrant accumulation of ER Ca(2+) release channels were detected in transgenic mutant A53T-αSyn mice. Collectively, these data establish a causative link between impaired ER Ca(2+) homeostasis and chronic ER stress in the degenerative cascades induced by mutant αSyn and suggest that Herp is essential for the resolution of ER stress through maintenance of ER Ca(2+) homeostasis. Our findings suggest a therapeutic potential in PD for agents that increase Herp levels or its ER Ca(2+)-stabilizing action.

Activating Transcription Factor 6 Limits Intracellular Accumulation of Mutant α1-Antitrypsin Z and Mitochondrial Damage in Hepatoma Cells

α(1)-Antitrypsin is a serine protease inhibitor secreted by hepatocytes. A variant of α(1)-antitrypsin with an E342K (Z) mutation (ATZ) has propensity to form polymers, is retained in the endoplasmic reticulum (ER), is degraded by both ER-associated degradation and autophagy, and causes hepatocyte loss. Constant features in hepatocytes of PiZZ individuals and in PiZ transgenic mice expressing ATZ are the formation of membrane-limited globular inclusions containing ATZ and mitochondrial damage. Expression of ATZ in the liver does not induce the unfolded protein response (UPR), a protective mechanism aimed to maintain ER homeostasis in the face of an increased load of proteins. Here we found that in hepatoma cells the ER E3 ligase HRD1 functioned to degrade most of the ATZ before globular inclusions are formed. Activation of the activating transcription factor 6 (ATF6) branch of the UPR by expression of spliced ATF6(1-373) decreased intracellular accumulation of ATZ and the formation of globular inclusions by a pathway that required HRD1 and the proteasome. Expression of ATF6(1-373) in ATZ-expressing hepatoma cells did not induce autophagy and increased the level of the proapoptotic factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) but did not lead to apoptotic DNA fragmentation. Expression of ATF6(1-373) did not cause inhibition of protein synthesis and prevented mitochondrial damage induced by ATZ expression. It was concluded that activation of the ATF6 pathway of the UPR limits ATZ-dependent cell toxicity by selectively promoting ER-associated degradation of ATZ and is thereby a potential target to prevent hepatocyte loss in addition to autophagy-enhancing drugs.

PKA phosphorylation couples hepatic inositol-requiring enzyme 1α to glucagon signaling in glucose metabolism

The endoplasmic reticulum (ER)-resident protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) is activated through transautophosphorylation in response to protein folding overload in the ER lumen and maintains ER homeostasis by triggering a key branch of the unfolded protein response. Here we show that mammalian IRE1α in liver cells is also phosphorylated by a kinase other than itself in response to metabolic stimuli. Glucagon-stimulated protein kinase PKA, which in turn phosphorylated IRE1α at Ser(724), a highly conserved site within the kinase activation domain. Blocking Ser(724) phosphorylation impaired the ability of IRE1α to augment the up-regulation by glucagon signaling of the expression of gluconeogenic genes. Moreover, hepatic IRE1α was highly phosphorylated at Ser(724) by PKA in mice with obesity, and silencing hepatic IRE1α markedly reduced hyperglycemia and glucose intolerance. Hence, these results suggest that IRE1α integrates signals from both the ER lumen and the cytoplasm in the liver and is coupled to the glucagon signaling in the regulation of glucose metabolism.

Endoplasmic reticulum homeostasis is crucial for in vitro blastocyst development

GRP78 (a 78-kDa glucose regulated protein) and its active downstream protein sXBP-1 are vital modulators of endoplasmic reticulum (ER) homeostasis and unfolded protein response. GRP78 deficient mouse embryos die in peri-implantation period. Higher level of sXBP-1 in degenerating embryos compared to healthy ones leads us to hypothesize that modulation of ER stress is crucial for preimplantation embryo development (PED). Hence, the physiopathologic impact of short-term and long-term activation of ER stress in PED was studied. Prospective study. 2-cell embryos, harvested from superovulated C57BL/6 mice were randomly divided into 4 groups. 1) Vehicle (control), 2) long-term tunicamycin (TUN; ER stress inducer; 0.5 μg/ml for 72h), 3) short-term TUN (0.5 μg/ml for 4h), 4) cycloheximide (protein synthesis inhibitor; 10 μg/ml) treatments. Embryos were morphologically assessed until day 4, evaluated for apoptosis and GRP78, XBP-1 and sXBP-1 were detected using RT-PCR. Cycloheximide caused developmental arrest at 2-cell stage all the time. Control, short- and long-term TUN-treated embryos were developed to compaction stage without any difference. However, number of embryos at blastocyst stage was significantly different among the control (92%), short- (39%) and long-term (2%) TUN-treated groups (P<0.001). RT-PCR analysis revealed significantly higher level of sXBP-1 mRNA in embryos treated with short-term (2-fold) and long-term (30-fold) TUN compared to that in control embryos. Similarly, GRP78 mRNA was significantly increased with TUN treatment compared to control group (P<0.01). Apoptotic cells in the blastocyst were enhanced with the TUN treatment too. Our results suggest that factors agitating embryonic ER homeostasis during in vitro development reduce radically blastocyst development. The differences in the rates of blastocyst development and GRP78 and sXPB-1 levels among the groups emphasize the importance of maintaining ER homeostasis on blastocyst development.

A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.

The Unfolded Protein Response Mediates Adaptation to Exercise in Skeletal Muscle through a PGC-1α/ATF6α Complex

Exercise has been shown to be effective for treating obesity and type 2 diabetes. However, the molecular mechanisms for adaptation to exercise training are not fully understood. Endoplasmic reticulum (ER) stress has been linked to metabolic dysfunction. Here we show that the unfolded protein response (UPR), an adaptive response pathway that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coactivator PGC-1α, which regulates several exercise-associated aspects of skeletal muscle function, mediates the UPR in myotubes and skeletal muscle through coactivation of ATF6α. Efficient recovery from acute exercise is compromised in ATF6α(-/-) mice. Blocking ER-stress-related cell death via deletion of CHOP partially rescues the exercise intolerance phenotype in muscle-specific PGC-1α KO mice. These findings suggest that modulation of the UPR through PGC1α represents an alternative avenue to improve skeletal muscle function and achieve metabolic benefits.

Bortezomib suppresses function and survival of plasmacytoid dendritic cells by targeting intracellular trafficking of Toll-like receptors and endoplasmic reticulum homeostasis

AbstractDendritic cells (DCs) play a pivotal role in the pathogenesis of inflammatory disorders, so suppressing the activity of DCs is instrumental in treating such diseases. In the present study, we show that a proteasome inhibitor, bortezomib, suppresses the survival and immunostimulatory function of human plasmacytoid DCs (pDCs) by targeting 2 critical points, intracellular trafficking of nucleic acid-sensingToll-like receptors (TLRs) and endoplasmic reticulum (ER) homeostasis. Among the immune cells in blood, pDCs were the most susceptible to the killing effect of bortezomib. This correlates with a decrease in the spliced form of a transcription factor XBP1, which rescues cells from apoptosis by maintaining ER homeostasis. Bortezomib suppressed the production of interferon-α and interleukin-6 by pDCs activated with a TLR9-stimulating CpG DNA and a TLR7-stimulating influenza virus, which appears to be partially independent of apoptosis. Bortezomib inhibited translocation of TLR9 from the ER to endolysosomes but not of an ER membrane protein, Unc93B1, that delivers TLR9 to endolysosomes. Thus, bortezomib suppresses the activity of pDCs by inhibiting intracellular trafficking of TLRs through disrupting the coordinated translocation of TLRs and Unc93B1 and by disturbing ER homeostasis. This study suggests that proteasome inhibitors may alleviate inflammatory disorders such as lupus and psoriasis that involve pDCs.

Endoplasmic Reticulum Stress As a Therapeutic Target in Cardiovascular Disease

Cardiovascular disease constitutes a major and increasing health burden in developed countries. Although treatments have progressed, the development of novel treatments for patients with cardiovascular diseases remains a major research goal. The endoplasmic reticulum (ER) is the cellular organelle in which protein folding, calcium homeostasis, and lipid biosynthesis occur. Stimuli such as oxidative stress, ischemic insult, disturbances in calcium homeostasis, and enhanced expression of normal and/or folding-defective proteins lead to the accumulation of unfolded proteins, a condition referred to as ER stress. ER stress triggers the unfolded protein response (UPR) to maintain ER homeostasis. The UPR involves a group of signal transduction pathways that ameliorate the accumulation of unfolded protein by increasing ER-resident chaperones, inhibiting protein translation and accelerating the degradation of unfolded proteins. The UPR is initially an adaptive response but, if unresolved, can lead to apoptotic cell death. Thus, the ER is now recognized as an important organelle in deciding cell life and death. There is compelling evidence that the adaptive and proapoptotic pathways of UPR play fundamental roles in the development and progression of cardiovascular diseases, including heart failure, ischemic heart diseases, and atherosclerosis. Thus, therapeutic interventions that target molecules of the UPR component and reduce ER stress will be promising strategies to treat cardiovascular diseases. In this review, we summarize the recent progress in understanding UPR signaling in cardiovascular disease and its related therapeutic potential. Future studies may clarify the most promising molecules to be investigated as targets for cardiovascular diseases.

Endoplasmic reticulum stress in β-cells and development of diabetes

The endoplasmic reticulum (ER) is a cellular compartment responsible for multiple important cellular functions including the biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. A myriad of pathological and physiological factors perturb ER function and cause dysregulation of ER homeostasis, leading to ER stress. ER stress elicits a signaling cascade to mitigate stress, the unfolded protein response (UPR). As long as the UPR can relieve stress, cells can produce the proper amount of proteins and maintain ER homeostasis. If the UPR, however, fails to maintain ER homeostasis, cells will undergo apoptosis. Activation of the UPR is critical to the survival of insulin-producing pancreatic beta-cells with high secretory protein production. Any disruption of ER homeostasis in beta-cells can lead to cell death and contribute to the pathogenesis of diabetes. There are several models of ER-stress-mediated diabetes. In this review, we outline the underlying molecular mechanisms of ER-stress-mediated beta-cell dysfunction and death during the progression of diabetes.

Cotranslational Targeting of XBP1 Protein to the Membrane Promotes Cytoplasmic Splicing of Its Own mRNA

Endoplasmic reticulum (ER) stress triggers the cytoplasmic splicing of XBP1 mRNA by the transmembrane endoribonuclease IRE1alpha, resulting in activation of the unfolded protein response, which maintains ER homeostasis. We show that the unspliced XBP1 (XBP1u) mRNA is localized to the membrane, although its product is neither a secretory nor a membrane protein and is released to the cytosol after splicing. Biochemical and mutagenic analyses demonstrated that membrane localization of XBP1u mRNA required its in-frame translation. An insertional frame-shift mutation greatly diminished both membrane localization and splicing of the XBP1u mRNA. Furthermore, membrane localization was compromised by puromycin treatment and required a hydrophobic region within XBP1u. These data demonstrate that the nascent XBP1u polypeptide recruits its own mRNA to the membrane. This system serves to enhance cytoplasmic splicing and could facilitate a more rapid response to ER stress, and represents a unique way of cotranslational protein targeting coupled to mRNA maturation.

Autophagy is essential for ER homeostasis and calcium mobilization in T lymphocytes (35.13)

Abstract The evolutionarily conserved intracellular bulk degradation pathway, macroautophagy, is involved in eliminating intracellular pathogens, endogenous antigen presentation in MHC-II pathway and T lymphocytes homeostasis. However, the underlying mechanisms by which autophagy regulates T lymphocyte function remain unknown. Here we used autophagy related protein 7 (Atg7) conditional knockout mice to investigate these mechanisms. Comparing to wild type T cells, Atg7-deficient T cells displayed impaired calcium influx upon TCR triggering or lower concentration of ionomycin stimulation. However, the proximal signaling of TCR in Atg7-deficient T cells was intact. The calcium influx defect in Atg7-deficient T cells may be related to an abnormal expansion of endoplasmic reticulum (ER) and higher intracellular calcium stores. We found that higher amount of ionomycin or the ER sarco/endoplasmic reticulum Ca2+ATPase (SERCA) pump inhibitor, thapsigargin, could rescue the calcium influx defect in Atg7-deficient T lymphocytes. Atg7-deficient T cells failed to efficiently proliferate and were blocked at S phase when stimulated by TCR or PMA plus ionomycin respectively. In contrast, Atg7-deficient T cells were fully activated and capable of secreting IL-2. Our results suggest that an important role for autophagy in T cells is to maintain ER homeostasis.

Lighting Up the Stressed ER

Balancing the capacity for protein maturation with changes in protein flux through the endoplasmic reticulum (ER) is crucial for maintaining ER homeostasis. In this issue, Merksamer et al. (2008) exploit a redox-sensitive fluorescent protein to monitor the environment inside the ER of living yeast, illuminating how this organelle responds to different perturbations.

The Unfolded Protein Response Regulator GRP78/BiP as a Novel Target for Increasing Chemosensitivity in Malignant Gliomas

Poor chemosensitivity and the development of chemoresistance remain major obstacles to successful chemotherapy of malignant gliomas. GRP78 is a key regulator of the unfolded protein response (UPR). As a Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER), GRP78 maintains ER homeostasis, suppresses stress-induced apoptosis, and controls UPR signaling. We report here that GRP78 is expressed at low levels in normal adult brain, but is significantly elevated in malignant glioma specimens and human malignant glioma cell lines, correlating with their rate of proliferation. Down-regulation of GRP78 by small interfering RNA leads to a slowdown in glioma cell growth. Our studies further reveal that temozolomide, the chemotherapeutic agent of choice for treatment of malignant gliomas, leads to induction of CHOP, a major proapoptotic arm of the UPR. Knockdown of GRP78 in glioblastoma cell lines induces CHOP and activates caspase-7 in temozolomide-treated cells. Colony survival assays further establish that knockdown of GRP78 lowers resistance of glioma cells to temozolomide, and, conversely, overexpression of GRP78 confers higher resistance. Knockdown of GRP78 also sensitizes glioma cells to 5-fluorouracil and CPT-11. Treatment of glioma cells with (-)-epigallocatechin gallate, which targets the ATP-binding domain of GRP78 and blocks its protective function, sensitizes glioma cells to temozolomide. These results identify a novel chemoresistance mechanism in malignant gliomas and show that combination of drugs capable of suppressing GRP78 with conventional agents such as temozolomide might represent a novel approach to eliminate residual tumor cells after surgery and increase the effectiveness of malignant glioma chemotherapy.