Mona Khoshbakht; Mohammad Mahdi Forghanifard; Hossein Aghamollaei; Jafar Amani
Abstract
Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining ...
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Background: Developing effective targeted treatment approaches to overcome drug resistance remains a crucial goal in cancer research. Immunotoxins have dual functionality in cancer detection and targeted therapy.Objective: This study aimed to engineer a recombinant chimeric fusion protein by combining a nanobody-targeting domain with an exotoxin effector domain. The chimeric protein was designed to bind surface-expressed GRP78 on cancer cells, facilitating internalization and inducing apoptosis to inhibit proliferation and survival.Methods: Using a flexible linker, we designed two constructs linking VHH nanobody domains to Pseudomonas exotoxin (PE) domains II, III, and Ib. These constructs were then optimized for expression in E. coli BL21 (DE3) using the pET28a vector. Following the expression of the recombinant proteins, we purified them and tested their binding capability, cytotoxicity, and ability to induce apoptosis in breast cancer cell lines MDA-MB-231 and MCF-7, as well as in control cell lines HEK-293 and MDA-MB-468. The binding affinity was measured using a cell-based ELISA, internalization was assessed through Western blotting, cytotoxicity was evaluated by an MTT assay, and apoptosis was determined using flow cytometry with an Annexin V kit.Results: The immunotoxin specifically bound to cancer cells expressing csGRP78. The results of the cytotoxicity test showed that the cytotoxic effect of two constructs, I and II, depended on concentration and time. With an increase in both components, the effect of recombinant proteins also increased. Both constructs were able to penetrate and induce apoptosis in csGRP78+ cells.Conclusion: These immunotoxin structures showed therapeutic potential against GRP78-expressing cancers, making them suitable candidates for targeted therapy pending in vivo studies.
Leila Rezanezhad; Jaleh Zolghadri; Behrouz Gharesi-Fard
Volume 10, Issue 4 , December 2013, , Pages 238-246
Abstract
Background: Preeclampsia (PE) is a pregnancy specific syndrome that is associated with high maternal and fetal morbidity and mortality. Glucose regulated protein78 (GRP78) is an Endoplasmic Reticulum (ER) protein which is expressed on the cell surfaces of trophoblast cells under stress or hypoxic condition. ...
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Background: Preeclampsia (PE) is a pregnancy specific syndrome that is associated with high maternal and fetal morbidity and mortality. Glucose regulated protein78 (GRP78) is an Endoplasmic Reticulum (ER) protein which is expressed on the cell surfaces of trophoblast cells under stress or hypoxic condition. GRP78 has a role in aggressive behavior of invasive cells and may play a role in normal placentation. Objectives: To investigate the autoantibody against GRP78 in the sera of patients with PE and to assess the correlation between antibody and severity of the disease. Methods: We evaluated the anti-GRP78 antibody within the sera of fifty pre-eclamptic (12 severe and 38 mild PE) and fifty healthy pregnant women using a home-made ELISA assay. Furthermore, western blot technique was used to assess the expression of GRP78 in placenta of healthy and pre-eclamptic women in their third trimester. The presence of anti-GRP78 antibody in the serum samples from pre-eclamptic and healthy women was also assessed. Results: GRP78 was expressed by placenta, and both healthy and preeclamptic women produced anti-GRP78 antibody. Although no significant difference was found between the pre-eclamptic and healthy women regarding the level of anti-GRP78 antibody, the difference between severe pre-eclamptic and healthy control women was statistically significant (p<0.003). Conclusion: The findings of the present study indicated that measurement of anti-GRP78 antibody may provide a new marker for severe pre-eclampsia. Yet, future studies are required to confirm this notion.
