The Antiproliferative And Apoptotic Potency Of Endoplasmic Reticulum-retained Anti-TfR Intrabody On Tumor Cells

Iron is an essential element for cell proliferation and metabolism. Transferring receptor (TfR)-mediated endocytosis is a major pathway for cellular iron uptake, and the TfR is highly overexpressed in malignant cells. This characteristic renders tumor cells more sensitive to iron depletion which is well known to cause cell cycle arrest and apoptosis. Intrabodies are defined as recombinant antibodies which are expressed intracellularly and addressed in specific subcellular compartments where they can bind, and consequently neutralize or modify the activity of their target antigens. In the field of cancer, intrabodies have been used to modulate the expression of proteins upregulated in tumors, such as erbB-2, IL-2 receptor, epidermal growth-factor receptor (EGFR), vascular endothelial growth factor receptor-2 (KDR), folate receptor, et al. The development of intracellularly expressed antibodies introduced a novel way of antigen neutralization causing a phenotypic or functional knockout.Objective: Aim to deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, in this study we attempt to intracellularly block surface expression of TfR by intracellular antibody technology, and consequently induce deprivation of iron uptake and affect the proliferation of tumor cells.Methods and results: we constructed two expression plasmid vectors coding for intracellular single-chain antibody against TfR extracellular domain with (scFv-HAK) or without (scFv-HA) endoplasmic reticulum (ER) retention signal, and then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Realtime-PCR assay indirectly indicates that iron uptake of scFv-HAK transfected cells was significantly decreased. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups.Conclusions: Our results presented here demonstrate that the intracellular expression of an anti-TfR single-chain antibody with ER retention signal dramatically inhibited the surface expression of TfR, by trapping the TfR and targeting it to the ER. Further, we show that retention of the receptor in the ER caused its function impaired and induced cell cycle G1 arrest and apoptosis, and consequently inhibits tumor cells proliferation. For the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.