Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (K_d=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Role of Prostate Apoptosis Response 4 in Translocation of GRP78 from the Endoplasmic Reticulum to the Cell Surface of Trophoblastic Cells

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) molecular chaperone that belongs to the heat shock protein 70 family. GRP78 is also present on the cell surface membrane of trophoblastic cells, where it is associated with invasive or fusion properties of these cells. Impaired mechanism of GRP78 relocation from ER to the cell surface was observed in preeclamptic cytotrophoblastic cells (CTB) and could take part in the pathogenesis of preeclampsia. In this study, we have investigated whether prostate apoptosis response 4 (Par-4), a protein identified as a partner of GRP78 relocation to the cell surface in prostate cancer cells, is present in trophoblastic cells and is involved in the translocation of GRP78 to the cell surface of CTB. Par-4 is indeed present in trophoblastic cells and its expression correlates with expression of membrane GRP78. Moreover, overexpression of Par-4 led to an increase of cell surface expression of GRP78 and decreased Par-4 gene expression reduced cell surface localization of GRP78 confirming a role of Par-4 in relocation of GRP78 from ER to the cell surface. Accordingly, invasive property was modified in these cells. In conclusion, we show that Par-4 is expressed in trophoblastic cells and is involved in transport of GRP78 to the cell surface and thus regulates invasive property of extravillous CTB.     

Suppression of Colorectal Cancer Liver Metastasis by Apolipoprotein(a) Kringle V in a Nude Mouse Model through the Induction of Apoptosis in Tumor-Associated Endothelial Cells

The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.     

Novel mechanism of anti-apoptotic function of 78-kDa glucose-regulated protein (GRP78): endocrine resistance factor in breast cancer, through release of B-cell lymphoma 2 (BCL-2) from BCL-2-interacting killer (BIK)

Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca(2+) from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.     

Procaspase-8失调与肿瘤细胞对TRAIL的耐受

肿瘤坏死因子相关凋亡诱导配体(TRAIL)可激活胱天蛋白酶（caspase）家族蛋白系列级联反应,最终诱导细胞凋亡. TRAIL选择性地诱导肿瘤细胞凋亡而不损伤正常细胞,使其成为治疗癌症的潜在药物靶点. 目前已知,细胞型FADD样白介素-1-β转换酶抑制蛋白(c FLIP)和凋亡抑制蛋白(IAPs)是肿瘤细胞对TRAIL耐受的主要原因.胱天蛋白酶原-8（procaspase-8）是TRAIL凋亡信号途径中的凋亡起始蛋白. 然而近年发现,在某些肿瘤细胞中procaspase-8功能失调常会阻碍凋亡信号传导,使肿瘤细胞对TRAIL诱导的凋亡产生耐受. 本文就其机制进行概述.     

Activation of multiple apoptotic pathways in human nasopharyngeal carcinoma cells by the prenylated isoflavone, osajin

Osajin is a prenylated isoflavone showing antitumor activity in different tumor cell lines. The underlying mechanism of osajin-induced cancer cell death is not clearly understood. In the present study, the mechanisms of osajin-induced cell death of human nasopharyngeal carcinoma (NPC) cells were explored. Osajin was found to significantly induce apoptosis of NPC cells in a dose- and time-dependent manner. Multiple molecular effects were observed during osajin treatment including a significant loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, enhanced expression of Fas ligand (FasL), suppression of glucose-regulated protein 78 kDa (GRP78), and activation of caspases-9, -8, -4 and -3. In addition, up-regulation of proapoptotic Bax protein and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, osajin induces apoptosis in human NPC cells through multiple apoptotic pathways, including the extrinsic death receptor pathway, and intrinsic pathways relying on mitochondria and endoplasmic reticulum stress. Thus, osajin could be developed as a new effective and chemopreventive compound for human NPC.     

Resveratrol induces apoptosis of human nasopharyngeal carcinoma cells via activation of multiple apoptotic pathways

Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been reported to trigger a variety of cancer cell types to apoptosis. Whether resveratrol shows any activity on human nasopharyngeal carcinoma (NPC) cells remained to be determined. The aim of this study was to investigate the effect and mechanism of resveratrol on human NPC cells. Treatment of resveratrol resulted in significant decrease in cell viability of NPC cell lines in a dose‐ and time‐dependent manner. A dose‐dependent apoptotic cell death was also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled resveratrol treatment resulted in a significant loss of mitochondrial transmembrane potential, release of cytochrome c, enhanced expression of Fas ligand (FasL), and suppression of glucose‐regulated protein 78 kDa (GRP78). These were followed by activation of caspases‐9, ‐8, ‐4, and ‐3, subsequently leading to DNA fragmentation and cell apoptosis. Furthermore, up‐regulation of proapoptotic Bax and down‐regulation of antiapoptotic Bcl‐2 protein were also observed. Taken together, resveratrol induces apoptosis in human NPC cells through regulation of multiple apoptotic pathways, including death receptor, mitochondria, and endoplasmic reticulum (ER) stress. Resveratrol can be developed as an effective compound for human NPC treatment. J. Cell. Physiol. 226: 720–728, 2011. © 2010 Wiley‐Liss, Inc.     

Modulation of the unfolded protein response in prostate cancer cells by antibody-directed against the carboxyl-terminal domain of GRP78

Receptor-recognized forms of α₂-macroglobulin (α₂M*) bind to cancer cell surface GRP78, which functions as a signaling receptor promoting proliferation and survival. Patients with prostate, ovary, and skin cancer may develop auto-antibodies to the α₂M* binding site which are receptor agonists whose presence indicates a poor prognosis. By contrast, antibodies directed against the COOH-terminal domain of GPR78 (anti-CTD antibody), are antagonists which down regulate pro-proliferative signaling and upregulate p53. Unfolded protein response (UPR) signaling plays an important role in cell survival and proliferation as well as apoptosis. We, therefore, studied the effect of anti-CTD antibody on UPR signaling in 1-LN and DU-145 prostate cancer cells. Treatment of these cells, which express GRP78 on their cell surface, with this antibody significantly downregulated IRE1-α, PERK, and ATF6α-dependent UPR signaling. By contrast, the pro-apoptotic protein GADD153 was elevated. Anti-CTD antibody treatment also elevated apoptotic components, cleaved PARP-1, and Erdj5. In general, a two to threefold effect was observed for the parameters which were studied. These studies suggest that anti-CTD antibody induces growth inhibitory and pro-apoptotic effects by modulating UPR signaling in human prostate cancer cells.     

The anticancer flavonoid chrysin induces the unfolded protein response in hepatoma cells

Chrysin is a natural and biologically active flavonoid with anticancer effects. However, little is known about the adaptive response of cancer cells to chrysin. Chrysin reportedly has proteasome inhibitor activity. Previous studies demonstrated that proteasome inhibitors might induce endoplasmic reticulum (ER) stress response. In this study, we aimed to determine the effects of chrysin on hepatoma cells and roles of the ER-resident protein GRP78 (glucose-regulated protein 78) in its action. Also, we investigated the effects of green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), a natural GRP78 inhibitor, on the sensitivity of hepatoma cells to chrysin. Here, we report that chrysin inhibits hepatoma cells growth and induces apoptosis in a dose-dependent manner. Chrysin induces GRP78 overexpression, X-box binding protein-1 splicing and eukaryotic initiation factor 2α phosphorylation, hallmarks of the unfolded protein response. GRP78 knockdown potentiates chrysin-induced caspase-7 cleavage in hepatoma cells and enhances chrysin-induced apoptosis. EGCG overcomes chrysin-induced GRP78 expression. Combination of EGCG potentiates chrysin-induced caspase-7 and poly (ADP-ribose) polymerase (PARP) cleavage. Finally, EGCG sensitizes hepatoma cells to chrysin through caspase-mediated apoptosis. These data suggest that chrysin triggers the unfolded protein response. Abrogation of GRP78 induction may improve the anticancer effects of chrysin. Combination of EGCG and chrysin represents a new regimen for cancer chemoprevention and therapeutics.     

Induction of endoplasmic reticulum stress in thymic lymphocytes by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period

Infection of thymic lymphocytes by a mink cell focus-forming murine leukemia virus induces apoptosis during the preleukemic period of lymphomagenesis. In this study, we observed that during this period, the viral envelope precursor polyprotein accumulated to high levels in thymic lymphocytes from mice inoculated with virus. Envelope accumulation occurred with the same kinetics as the induction of endoplasmic reticulum (ER) stress, which resulted in the upregulation of the 78-kDa glucose-regulated protein (GRP78). In thymic lymphomas, GRP78 levels were higher than those in virus-infected preleukemic cells, and GRP58 was upregulated. These results suggest that Env precursor accumulation induces ER stress, which participates in thymic lymphocyte apoptosis. The subsequent upregulation of ER chaperone proteins GRP78 and GRP58 may contribute to rescuing cells from virus-induced apoptosis.     

Chelation of Extracellular Calcium-Induced Cell Death was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

GRP78 and Cripto form a complex at the cell surface and collaborate to inhibit transforming growth factor beta signaling and enhance cell growth

Cripto is a multifunctional cell surface protein with important roles in vertebrate embryogenesis and the progression of human tumors. While Cripto has been shown to modulate multiple signaling pathways, its binding partners do not appear to fully explain its molecular actions. Therefore, we conducted a screen aimed at identifying novel Cripto-interacting proteins. This screen led to our identification of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone that is also expressed at the surfaces of tumor cells. Here we demonstrate that Cripto and GRP78 interact at the cell surfaces of multiple cell lines and that their interaction is independent of prior association within the ER. Interestingly, short hairpin RNA knockdown of endogenous GRP78 resulted in enhanced transforming growth factor β (TGF-β) signaling, indicating that like Cripto, GRP78 inhibits this pathway. We further show that when coexpressed, GRP78 and Cripto collaborate to antagonize TGF-β responses, including Smad phosphorylation and growth inhibition of prostate cancer cells grown under anchorage-dependent or -independent conditions. Finally, we provide evidence that cells coexpressing GRP78 and Cripto grow much more rapidly in soft agar than do cells expressing either protein individually. Together, our results indicate that these proteins bind at the cell surface to enhance tumor growth via the inhibition of TGF-β signaling.