Date of Completion
6-29-2015
Embargo Period
12-20-2015
Keywords
E-peptide, human Eb-peptide (hEb), lamellipodia outspread, integrin, clathrin-mediated endocytosis (CME), antimicrobial peptide (AMPs)
Major Advisor
Thomas T. Chen
Associate Advisor
Charles Giardina
Associate Advisor
Juliet Lee
Associate Advisor
Lawrence E. Hightower
Associate Advisor
Ping Zhang
Field of Study
Molecular and Cell Biology
Degree
Doctor of Philosophy
Open Access
Campus Access
Abstract
Insulin-like growth factor (IGF-I) is well known to play various significant roles in promoting growth, metabolism, differentiation, and neuroendocrine regulation in all vertebrates. The functions of E-domain peptides from pro-IGF-I, however, were overlooked. Some E-peptide isoforms are known to have IGF-I-like growth-promoting activity, and the ability to translocate into nucleus and nucleoli even with the presence of pre-pro hormone signal peptide. In our laboratory, several anti-cancer activities have been identified for the longest isoforms of human and rainbow trout E-peptides. The activities include dose-dependent inhibition of colony formation, inhibition of cancer cell metastasis and invasion through matrigel, suppression of cancer-induced angiogenesis, and attenuation of expression of apoptotic genes in favor of cell death. Here in this thesis, by utilizing a new double tagged expression construct, a recombinant hEb peptide with over 99 % purity was prepared. With its unusual high isoelectric point and the dimer formation characteristic through a single disulfide bond formation, hEb is able to adhere to negative charged 96-well culture plates rapidly, and pull down cell plasma membrane by interacting directly with at least the phospholipids of the cell membrane. Furthermore, hEb-peptide can trigger clathrin-mediated endocytosis. Coordinating among these actions, hEb-peptide enhanced highly metastatic anoikis resistant breast carcinoma cells (MDA-MB-231) to attach to substratum and induce lamellipodia outspread. On top of that, the amphiphilic hEb also exerts a rapid, non-apoptotic cancer cells killing activity through accumulation of hEb-peptide on the cell surface and disrupting the integrity of the plasma membrane, which resembles the action of some antimicrobial peptides and cell penetrating peptides. This killing activity is not only cell type specific but also peptide structure dependent. The cancer cell killing activity of hEb-peptide is manipulable through the presence of a 6x-His tag and transition metal cations such as Co2+. Results described in this thesis offer the opportunity to develop hEb-peptide as a controllable/locally activated therapeutic for human cancer management.
Recommended Citation
Yeh, Yang-Hui, "A Study on the Attachment and Lamellipodia Outspread in Human Breast Carcinoma Cells (MDA-MB-231) and Rapid Cancer Cells Killing Activity Induced by Recombinant Human E-peptide of Pro-IGF-I" (2015). Doctoral Dissertations. 804.
https://digitalcommons.lib.uconn.edu/dissertations/804
Movie 1_hEb endocytosis MDAMB231
Movie 2_hEb killing (9µM) SKNF1.mp4 (72740 kB)