ANTP‐SmacN7 fusion peptide‐induced radiosensitization in A549 cells and its potential mechanisms

Abstract

Background: Radioresistance in tumors limits the curative effect of the radiotherapy. Mimetic compounds of second mitochondria-derived activator of caspase (Smac) are potential new tumor radiation-sensitizing drugs because they can increase radiation-induced tumor cell apoptosis. Here, we observed the radiosensitization effect of a new Smac mimetic Antennapedia protein (ANTP)-SmacN7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells. Methods: The ANTP-SmacN7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells. A549 cells were divided into the control, radiation-only, ANTP-SmacN7-only and ANTP-SmacN7 + radiation groups. The cells were exposed to 0, 2, 4 and 6 Gy, with 20 μmol/L of ANTP-SmacN7. The radiation-sensitizing effects of the ANTP-SmacN7 fusion proteins were observed via clonogenic assay. Apoptosis was detected using flow cytometry. A comet assay was used to assess DNA damage. The levels and degrees of cytochrome-c, PARP, H2AX, caspase-8, caspase-3, and caspase-9 activation were detected via western blot assay. The radiation sensitization of the fusion peptide, expression of γ-H2AX and C-PARP were compared after adding the caspase inhibitor, Z-VAD. Results: ANTP-SmacN7 fusion proteins entered the cells and promoted A549 cell radiosensitization. Treatment with ANTP-SmacN7 + radiation significantly reduced the A549 cell clone-forming rate, increased the cytochrome-c, cleaved caspase-8, cleaved caspase-3 and cleaved caspase-9 expression levels, promoted caspase activation, and increased the rate of radiation-induced apoptosis. The ANTP-SmacN7 fusion peptide significantly increased radiation-induced doublestranded DNA rupture in the A549 cells and increased DNA damage. Adding Z-VAD reduced the fusion peptide's proapoptotic effect but not the level of double-stranded DNA breakage. Conclusions: The ANTP-SmacN7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells. This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.

Functionalization of biomedical materials using fusion peptides for tissue regeneration

“…Xie et al and Zhang et al combined the Drosophila Antennapedia protein (ANTP) with seven amino acid residues (SmacN7) at the amino terminus of the second mitochondria-derived activator of caspase (Smac) and demonstrated the effectiveness of the fusion peptide in improving tumor cell radiosensitivity. [39,62] Smac is the only known molecule that can directly inhibit apoptosis-inhibiting proteins in mammals [63]. SmacN7 is the smallest active protein of Smac, and can promote apoptosis responding to apoptotic stimulation [64].…”