Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A comparative in vitro study using EGFR-positive breast cancer cells
“…To investigate the reason for the enhanced activity of the SR mutant compared to WT, we analyzed the binding affinity of various EGF (RS, SR) and WT-EGF for EGFR expressed in A431 cells by means of in situ cell ELISA [ 28 ]. The equilibrium dissociation constant values (K d ) were evaluated using a nonlinear regression analysis.…”
Section: Resultsmentioning
confidence: 99%
“…The K d value revealed a 1.9-fold lower for SR compared with WT. Early study demonstrated decreased cytotoxicity for an EGF mutant with a lower binding affinity to EGFR in MDA-MB-468 (overexpressed EGFR) [ 28 ], therefore the increased affinity of the SR mutant toward EGFR is supposed to be involved in its increased growth inhibitory effect. Figure 9.…”
Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.
“…To investigate the reason for the enhanced activity of the SR mutant compared to WT, we analyzed the binding affinity of various EGF (RS, SR) and WT-EGF for EGFR expressed in A431 cells by means of in situ cell ELISA [ 28 ]. The equilibrium dissociation constant values (K d ) were evaluated using a nonlinear regression analysis.…”
Section: Resultsmentioning
confidence: 99%
“…The K d value revealed a 1.9-fold lower for SR compared with WT. Early study demonstrated decreased cytotoxicity for an EGF mutant with a lower binding affinity to EGFR in MDA-MB-468 (overexpressed EGFR) [ 28 ], therefore the increased affinity of the SR mutant toward EGFR is supposed to be involved in its increased growth inhibitory effect. Figure 9.…”
Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.
“…A small clinical study in patients with advanced bowel cancer, having sEGFR measured, used FOLFOX6 and gefitinib, demonstrating a more objective response in those with higher sEGFR [82]. Research studies directed to the management of EGFR [83], including new gene therapies [84], demonstrated the feasibility of the suppression of the Notch3 gene with reduced internalisation of EGFR [85], or the construction of mutant forms of the EGF ligand blocking this receptor [86], or the genetic silencing by small RNAs of interference (siRNA) against EGFR [87]. New therapies, new antibody formations or new associations with TKIs signal the importance of adequately measuring EGFR, both as a prognostic biomarker, and as a treatment predictor, in women with TN breast tumours.…”
Background: Epidermal growth factor receptor (EGFR) overexpression has been considered a poor prognostic factor in breast cancer.Methodology: A prospective study of 206 women with breast cancer analysed by stages (I, II, III and IV) and by immunohistochemical subtype (Luminal A, Luminal B, HER2+ and triple-negative (TN)); 89 healthy controls with normal recent mammography were included. The EGFR measured in the serum (sEGFR) was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) method (R&D Systems kit DY231) collected by blood before any treatment in patients. Kaplan-Meier method and Cox regression were carried out to obtain the prognostic value, considering significance if p < 0.05.Results: With a median follow-up of 36.6 months, 47 deaths occurred. Multivariable Cox regression showed difference of overall survival (OS) associated with sEGFR levels (sEGFR ≤ or > 47.8 ng/mL) in patients with TN cancers, but not of Luminal A, Luminal B or HER2+ subtypes; adjusted by stage, the death risk increased by approximately 415% [hazard ratio (HR): 5.149 (1.900-13.955), p = 0.001] for patients with sEGFR > 47.8 ng/mL compared to patients with a lower sEGFR value. There was no significant correlation of sEGFR with staging, histological tumour grade (G1/ G2/G3), Ki67 (< or ≥14%) or body mass index.Conclusions: Increased sEGFR expression in patients with TN tumours is a significant predictor of lower OS and its quantification is inexpensive and straightforward.
“…8a and Additional file 8). In particular, the QYRD sequence was spotted to participate in EGF interaction with the receptor by sitedirected mutagenesis [49] and molecular dynamics [50].…”
Section: Analysis Of the Putative Rnase3-egfr Interaction By Moleculamentioning
The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory “core-response” independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners.
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