Annat Raiter

Activation of GRP78 on Endothelial Cell Membranes by an ADAM15-Derived Peptide Induces Angiogenesis

Impaired angiogenesis is one of the features of ischemic diseases. We have previously identified, by screening a phage display peptide library, a peptide that induces angiogenesis in endothelial cells under hypoxic conditions by binding the cell’s membrane heat shock protein GRP78. Protein data base search identified 4 amino acids (HWRR) of that synthetic peptide present on the ADAM15 metalloprotease domain, a protein considered to be involved in neovascularization. Three peptides were synthesized according to the ADAM15 sequence placing HWRR at different positions. Peptide ADoPep1 exhibited significant angiogenic properties under hypoxic conditions as determined by cell proliferation, migration and tube formation. In a mouse hind limb ischemia model, a single injection of the peptide restored blood perfusion. The identified peptide was found to activate GRP78 on endothelial cell membrane and siRNA directed against the GRP78 mRNA interfered with induction of angiogenesis by the peptide. The peptide binding induced a decrease in heat shock protein GRP78 that is overexpressed under hypoxic conditions. The mechanism of peptide-induced angiogenic activity involves inhibition of apoptosis as well as increased Akt phosphorylation and ERK 1/2 activation. The peptide did not induce VEGF receptor-2 protein synthesis and phosphorylation, suggesting a VEGF-independent mechanism of angiogenesis.

Peptide-binding GRP78 protects neurons from hypoxia-induced apoptosis

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21xa0days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.

Colon cancer cells expressing cell surface GRP78 as a marker for reduced tumorigenicity

BackgroundThe glucose regulated heat shock protein 78 (GRP78) is a central regulator of ER (endoplasmic reticulum) stress due to its pro-survival property. Up regulated GRP78 expression in tumor cells has been correlated with aggressive malignancies whereas some reports have predicted an improved prognosis. Over-expression of GRP78 in the ER promotes its localization to the cell surface on several cell types including tumor cells.MethodsIn order to elucidate whether GRP78 receptor positive and negative tumor cells manifest different properties in colorectal cancer, we first artificially separated GRP78 positive and negative sub-populations from HM7 and HCT116 cell lines using anti GRP78 antibody coated magnetic beads.ResultsOnly GRP78 negative cells were highly proliferative, induced significant growth in tumor size in nude mice and metastasized to the liver in a human metastatic colorectal carcinoma model in mice. In contrast, GRP78 positive cells manifested reduced proliferation, colony formation, tumor growth and liver metastases. The reduced tumorigenicity of GRP78 positive subpopulation was abrogated by silencing GRP78 expression using siRNA oligomers. In our efforts to induce cell surface GRP78, we subjected the cells to doxorubicin and taxol that increased significantly the percent of GRP78 positive population. Cells pre-incubated with doxorubicin exhibited reduced proliferation and tumor growth in mice.ConclusionThis study demonstrates the significance of cell surface GRP78 in colon cancer, which may be used as a marker for reduced tumorigenicity.

Endothelial cell surface vimentin binding peptide induces angiogenesis under hypoxic/ischemic conditions.

We have previously identified several angiogenic peptides that bind cell surface proteins by screening a phage display peptide library on human umbilical endothelial cells exposed to hypoxic conditions. In this study we describe one of the selected peptides, SP. We found by protein precipitation of endothelial cell lysates that the 12 amino acid SP peptide binds cell surface vimentin. Surprisingly, vimentin was detected on the cell surface of about 30% of intact endothelial cells under both normoxic and hypoxic conditions, as was demonstrated by fluorocytometric analysis on viable cells. The assessment of SP in the induction of angiogenesis was established by a significant increase in endothelial cell proliferation and tube formation under hypoxic conditions and not under normoxic conditions. Cell proliferation and tube length increased two-fold in endothelial cells in the presence of 10 ng/ml SP peptide when compared to controls. The specificity of SP binding to vimentin was demonstrated by SP inhibition of anti-vimentin binding and by the inhibition of tube formation in cells transfected with siRNA against vimentin. Local intramuscular administrations of the peptide SP to ischemic hind limbs using the mouse hind limb ischemia model, demonstrated that SP inoculated at 1 and 10 μg, improved blood perfusion compared to inoculations with an irrelevant peptide or PBS. The recovery of blood perfusion correlated with the increase in the number of detectable capillaries in the ischemic limb. The development of novel peptides for the induction of pro-angiogenic activity may pave the way for new therapeutic strategies in the treatment of cardiovascular ischemic diseases.

Cell surface GRP78: A potential marker of good prognosis and response to chemotherapy in breast cancer.

The 78-kDa glucose-regulated protein (GRP78) is a stress induced heat shock protein which, under limiting conditions, functions as a cell surface signaling receptor. Tumor cells are considered to be subjected to a physiologically stressful microenvironment due to their excessive growth. The role of GRP78 in tumor survival has been of notable interest. The present study aimed to assess the potential prognostic and predictive value of cell surface GRP78 expression in breast cancer tumor cells. Cell surface and cytoplasmic expression of GRP78 was examined by immunohistochemical staining of GRP78 in breast cancer archival paraffin-embedded tumor specimens. The cohort studied included breast cancer patients with operable T1,2, estrogen receptor-positive, node-negative cancer who were assessed using the Oncotype DX gene profile, as well as patients with locally advanced disease prior to and following neoadjuvant systemic treatment. GRP78 values were compared between the 2 groups, and prior to and following systemic treatment. Association analyses between GRP78 expression and prognostic markers were also performed. Cox regression analysis was used to examine the impact of these variables on disease-free survival (DFS). No differences in cytoplasmic GRP78 expression were observed. By contrast, the rates of cell surface GRP78 expression were 74.1% in the early stage operable patients, 36% in neoadjuvant systemic treatment patients prior to treatment and 62.5% in patients following systemic treatment (P<0.039). Positive cell surface GRP78 expression was associated with increased expression of the progesterone receptor (P=0.024), p53 expression (P=0.022) and improved DFS (P=0.047). In the case of GRP78 positivity, a trend for a superior response to chemotherapy was observed (P=0.19). The results of the present study indicated that cell surface GRP78 may be used as a marker for good prognosis in breast cancer and a potential marker for response to chemotherapy.

The presence of anti-GRP78 antibodies in the serum of patients with colorectal carcinoma: a potential biomarker for early cancer detection.

Background The identification of new biomarkers is required for early diagnosis of colorectal carcinoma patients (CRC), since about 20% of these patients are initially diagnosed with a distant metastatic disease. GRP78, a heat shock protein, functions also as a cell surface signaling receptor of cells under physiological stress. GRP78 was found to be expressed on the cell surface of various tumor cells. The presence of autoantibodies to GRP78 in cancer patients serum was found to be correlated with a poor prognosis. In this study we aimed to identify anti-GRP78 antibodies in the serum of 85 patients diagnosed by colonoscopy, as an early detection biomarker. Methods We developed an ELISA assay with recombinant GRP78 immobilized on 96-well culture plates and used an anti-IgG antibody to measure the sole anti-GRP78 IgGs. Results Testing for anti-GRP78 showed a significant increase in antibody titer in patients with a polyp and in CRC patients (p<0.001) compared to healthy subjects. Conclusions This is the first study showing the presence of anti-GRP78 at the very early stages of CRC.

Peptide Binding Glucose Regulated Protein 78 Improves Type 1 Diabetes by Preventing Pancreatic β Cell Apoptosis

BACKGROUND AND OBJECTIVESnDiabetes Type 1 is characterized by hyperglycemia due to reduced insulin secretion that results from the death of pancreatic β cells. It was suggested that endoplasmic reticulum (ER) stress is associated with the autoimmune-mediated β cell destruction. Glucose regulated protein 78 (GRP78) functions as a key regulator to maintain the ER function. Under stress conditions GRP78 is up-regulated and expressed on the cell surface serving as a signaling receptor. Our first objective was to examine the effects of peptide binding cell surface GRP78 to reduce the deleterious effects of diabetes induced by streptozotocin. The second objective was to demonstrate the ability of the peptide to protect the pancreatic β cells from apoptosis.nnnMETHODSnThe effect of ADoPep on weight loss, HbA1c levels and anti GRP78 antibody titers was evaluated in a diabetes mouse model. The effect of ADoPep on the pancreatic β Ins1E cell apoptosis was determined by FACS analysis.nnnRESULTSnThe administration of ADoPep to diabetic mice retained the weight loss and reduced HbA1c significantly in 60% of mice. Titers of anti GRP78 antibodies increased in 70% of the treated mice. Apoptosis was significantly inhibited in stressed pancreatic β Ins 1E cells.nnnCONCLUSIONSnWe demonstrate that administration of the peptide ADoPep to diabetic mice improved type 1 diabetes by preventing pancreatic β cell apoptosis.