Antitumoral properties of epidermal growth factor derivatives

The members of the epidermal growth factor (EGF) / ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this thesis work we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. The lack of EGFR-mediated growth-stimulatory activity prompted us to evaluate EGFt for targeted delivery of 111In, an Auger electron emitter, into EGFR-positive cancer cells. An 111In-DTPA-EGFt radioconjugate was developed and its properties were analyzed and compared to those of 111In-DTPA-hEGF. First we determined that 111In-DTPA-EGFt displays high specificity and affinity for EGFR. However, the cellular uptake of 111In-DTPA-EGFt resulted to be lower than that of 111In-DTPA-hEGF. Once internalized, 111In-DTPA-EGFt showed a high efficiency to accumulate into the cell nucleus, where the radioactivity emitted by 111In may damage the DNA. In accordance, 111In-DTPA-EGFt showed to be cytotoxic in vitro against breast cancer cells, although its cytotoxicity was lower compared to 111In-DTPA-hEGF. In vivo studies revealed a longer half-life in blood for 111In-DTPA-EGFt than for 111In-DTPA-hEGF and higher uptake in the kidney, with minor accumulation in other normal tissues. 111In-DTPA-EGFt accumulated in MDA-MB-468 tumors where, interestingly, 111In-DTPA-EGFt was detected in a great proportion in the cell nucleus. All the data obtained from this work indicate that EGFt may be a potential EGFR blocker for cancer therapy and also an attractive ligand for delivery of cytotoxic agents into the nucleus of EGFR-positive cancer cells. ​
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