Abstract
IntroductionThe F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy.MethodsF3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks.ResultsMembrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023).Conclusion111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation.
Highlights
The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin
The tumor-homing peptide F3, a 31-mer peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), derived from human high mobility group protein 2 (HMGN2) has been shown to bind to nucleolin expressed on the membrane of cancer cells, neovasculature, and endothelium
Of the internalized fraction of 111In-benzyl-diethylenetriaminepentaacetic acid (BnDTPA)-F3, 37% translocated to the nuclei, similar to the 33.6% found for fluorescein isothiocyanate (FITC)-F3
Summary
The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy. Targeted radiotherapy uses alpha-, beta-, or Auger electron emissions from radionuclides to irradiate cancer cells and cause tumor growth arrest. Cellular internalization and subsequent nuclear localization are deemed necessary for Auger electron radiation to be effective for cancer therapy [8]. The tumor-homing peptide F3, a 31-mer peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), derived from human high mobility group protein 2 (HMGN2) has been shown to bind to nucleolin expressed on the membrane of cancer cells, neovasculature, and endothelium. A F3-peptide dimer labeled with the alpha-emitter 213Bi (213Bi-(F3)2) for molecular radiotherapy of membranar-nucleolin-expressing MDA-MB-435 cells was shown to be effective in vitro and in xenografts in mice [19]. When F3 was labeled with 67Ga, it accumulated in MDA-MB-435 tumor xenografts in vivo, as shown by PET imaging [19]
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