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Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human...
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... H125 cells in the pres- ence of AG1478 and in the absence of exogenously added EGF reduced the growth rate of the cells to about 69% in a concentra- tion as low as 0.1 ,IM and to 3% at I ,UM ( Figure 2B). In contrast, H661 NSCLC cells, which have undetectable EGFR levels (Table 1), are resistant to treatment with AG1468, indicating that the effect of H125 is specific and non-toxic. Identical results were obtained with another EGFR tyrosine kinase inhibitor, AG1517 [PD 153035 (Fry et al, 1994)] ( Figure 2B). ...
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... It has been reported that cholesterol depletion of the plasma membrane by methylβ-cyclodextrin causes ligand-independent activation of the epidermal growth factor (EGF) receptor (Chen and Resh 2002;Ringerike et al. 2002). The ganglioside GM3 is also known to modulate EGF receptor activity (Bremer et al. 1986;Meuillet et al. 1999Meuillet et al. , 2000, either directly (Rebbaa et al. 1996) or via a phosphatase (Suarez Pestana et al. 1997). Changes in GM3 content do not affect EGF binding, but GM3 depletion increases EGF receptor autophosphorylation, whereas GM3 enrichment decreases EGF receptor autophosphorylation (Meuillet et al. 1999(Meuillet et al. , 2000. ...
Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.
... In keratinocytes, GM3, but not GM1 or GD1a, stimulates proliferation (Paller et al., 1993), which contrasts with the situation in K562 leukemia cells, where GM3 inhibits growth, while GM1, GM2 or GD1a have no effect (Nakamura et al., 1991). In lung adenocarcinoma cells, GM3 inhibits the proliferative action of EGF, most probably by activation of a EGF-receptor-directed phosphotyrosine phosphatase (Suarez Pestana et al., 1997). Inhibition by gangliosides of T-cell proliferation seems also to result from the activation of a protein phosphatase, in this case resulting in a diminished phosphorylation of the retinoblastoma protein (Irani, 1998). ...
... Previous results from Ferris and colleagues have suggested a dependence on EGFR density for cetuximab-mediated HLA class I modulation (Srivastava et al., 2015). Consequently, we selected two human tumor cell lines having high to intermedium EGFR expression levels, A431 that express 2-3 × 10 6 EGFR molecules per cell (Haigler et al., 1978) and H125 that express 2.1 × 10 5 EGFR molecules per cell (Suarez Pestana et al., 1997). U1906 cell line which does not express the EGFR at all (Suarez Pestana et al., 1997) was used as negative control. ...
... Consequently, we selected two human tumor cell lines having high to intermedium EGFR expression levels, A431 that express 2-3 × 10 6 EGFR molecules per cell (Haigler et al., 1978) and H125 that express 2.1 × 10 5 EGFR molecules per cell (Suarez Pestana et al., 1997). U1906 cell line which does not express the EGFR at all (Suarez Pestana et al., 1997) was used as negative control. First, we examined how EGFR signaling impacts HLA class I membrane expression in these tumor cell lines. ...
Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor – T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8⁺ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.
... It was corroborated by Western blot that A431 and MDA-MB-468 cells express high levels of EGFR, which were moderate in the case of H125 cells and low for PC-3 cells. Previous reports demonstrated that A431 cells and MDA-MB-468 tumor cells express 2,6 × 10 6 and 1.3 × 10 6 EGFR molecules per cell, respectively [22,23], while H125 cells express 2.1 × 10 5 receptors [24] and PC-3 cells only ~3 × 10 4 [25]. We also show that nimotuzumab has de capacity to recognize the EGFR in three out of the five tumor cell lines tested, but neither in PC3 cells which display low levels of the receptor, nor in U1906, this last cell line reported with negative expression of the receptor. ...
... GM3 interacts with basal membrane components, including adhesion molecules, to regulate cell adhesion and migration and GM3 directly interacts with GFRs, including EGFR, fibroblast growth factor receptor, platelet-derived growth factor receptor, vascular endothelial growth factor and insulin receptor, to modulate the receptor function and subsequently affect the intracellular signaling pathways (20)(21)(22)(23). GM3 also indirectly regulates GFR activity by affecting the interaction between GFR and membrane proteins, including integrins, CD9 and CD82 tetraspanins (24)(25)(26) or by affecting the intracellular Src kinase and protein tyrosine phosphatase activity (27)(28)(29). GM3 also regulates the activation of non-tyrosine kinase receptors, including G-protein coupled receptors (30). Thus, the mechanisms by which GM3 function are particularly complicated. ...
The ganglioside GM3 exerts its different effects via various growth factor receptors. The present study investigated and comparatively analyzed the opposing effects exerted by GM3 on the migration of mouse hepatocellular carcinoma Hepa1‑6 cells via epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet). The results demonstrated that GM3 inhibited EGF‑stimulated motility, but promoted HGF‑stimulated motility of the Hepa1‑6 cells via phosphatidylinositol 3‑kinase/Akt‑mediated migration signaling. It is well established that the main cytokines modulating cell proliferation, invasion and metastasis are different in different types of tumor. This difference may, at least in part, explain why GM3 exerted its actions in a tumor‑type specific manner.
... GM3 can directly influence the activity of EGFR by interaction with N-linked GlcNAc termini of the receptor [Yoon et al., 2006]. GM3 also indirectly regulates EGFR activity by influencing the interaction between EGFR and membrane proteins such as integrins and CD9 [Ono et al., 2001;Kawakami et al., 2002] or by affecting the intracellular Src kinase and protein tyrosine phosphatase activity [Suarez Pestana et al., 1997;Biscardi et al., 1999]. In this paper, we only investigated the role of EGFR. ...
Two related sublines derived from murine ascites hepatoma cell lines Hca-F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be differ in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3-suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
... Conversely, direct exogenous administration of GM3 results in inhibition of EGFR function [62], likely due to inhibitory effects on receptor autophosphorylation. It has been demonstrated in a number of tumors that GM3 exerts its effect on EGFR though the activation of a tyrosine phosphatase [63]. ...
The oligosaccharide chains, or glycans, that decorate cell surface glycoproteins and glycolipids are among the most complex and diverse structures in vertebrate cells. It is estimated the well over half of all human proteins are glycosylated. Their expression is exquisitely regulated and is the result of the coordinated activity of distinct glycosyltransferases and glycosyl hydrolases that add or remove individual sugars to complete each glycan chain. Aberrantly expressed cell surface glycoconjugates are associated with malignant transformation, tumor progression, and metastasis and are predominantly the result of alterations in their biosynthetic machinery. They mediate key pathophysiological events during tumorigenesis including altered cellular adhesion and invasivity, molecular trafficking, receptor activation, and intracellular signal transduction in tumors.
... Nimotuzumab is a humanized version of the hR3 antibody developed in Havana, Cuba [36,37], which is used in cancer therapy outside of the US and EU. Its reduced side effects are attributed to its lower affinity resulting in improved pharmacokinetics with tumor-specific enrichment without significant binding to EGFR-expressing epithelial cells in the skin. ...
Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.
... While mean of fluorescence intensity (MFI) was examined (Figure 1b), the saturating mAb concentration was of 10–20 µg/mL in both cell lines. As reported before, A 431 showed a higher antigen density [11] on cell surface than NCI-H125 cell line [12]. In the subsequent experiments we always used the parameter % of binding for the analysis because demonstrate less variability of the results when the assay is performed multiple times (RSD less than 1% at 3 µg/mL of mAb). ...
An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use.
... Similar results were obtained when surface EGFR expression was analyzed (Fig. 1B). These results are in agreement with previous reports showing that the A431 cells express 2-3 x 10 6 EGFR molecules per cell; 32 the H125 and U1810 cells express 2.1 x 10 5 and 7 x 10 3 EGFR molecules per cell, respectively, 33 whereas the MDA-MB-231 cells have a weak expression, 34 and the U1906 cell line does not express the EGFR at all. 33 The different cell lines were incubated with increasing concentrations of nimotuzumab, cetuximab, or their Fabs. ...
... These results are in agreement with previous reports showing that the A431 cells express 2-3 x 10 6 EGFR molecules per cell; 32 the H125 and U1810 cells express 2.1 x 10 5 and 7 x 10 3 EGFR molecules per cell, respectively, 33 whereas the MDA-MB-231 cells have a weak expression, 34 and the U1906 cell line does not express the EGFR at all. 33 The different cell lines were incubated with increasing concentrations of nimotuzumab, cetuximab, or their Fabs. The ratios between the nimotuzumab and cetuximab binding capabilities were measured by flow cytometry. ...
Nimotuzumab is an EGFR-targeting antibody that has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. We investigated whether different clinical behavior of nimotuzumab is related to its bivalent/monovalent binding profile. Binding properties of nimotuzumab and cetuximab, the most development of anti-EGFR antibodies, were studied in vitro using chip surfaces and cells with varying EGFR expression levels. Experimental observations demonstrated that in contrast to cetuximab, the intrinsic properties of nimotuzumab required bivalent binding for stable attachment to the cellular surface, leading to nimotuzumab selectively binding to cells that express moderate to high EGFR expression levels. At these conditions, both antibodies bound bivalently, and accumulated to similar degrees. When EGFR density is low, nimotuzumab monovalent interaction was transient, whereas cetuximab continued to interact strongly with the receptors. We compared the in vitro anti-tumor efficacy of nimotuzumab and cetuximab. Cetuximab decreased the cell viability and induced apoptosis for all the tested cell lines, effects which did not depend on EGFR expression level. In contrast, nimotuzumab also provoked significant anti-cellular effects, but its anti-tumor capacity decreased together with EGFR expression level. Cetuximab Fab fragment was able to impact tumor cell survival, whereas nimotuzumab fragment totally lost this effect. Tumor-xenograft experiments using cells with a high EGFR expression revealed similar tumor growth inhibiting effects for both antibodies. This study suggests an explanation for nimotuzumab clinical profile, whereby anti-tumor activity is obtained in absence of severe toxicities due to its properties of bivalent binding to EGFR.
