Monitoring the correlation between I-uptake and apoptosis using Apoptosis-targeting peptide-1 (ApoPep-1)

Objectives Radionuclide therapy using I-131 with Sodium Iodide Symporter (NIS) system induces apoptosis in NIS-expressing tumor. In this study, the correlation between the amount of I-uptake and apoptosis was monitored to evaluate the therapeutic efficacy of radio-iodide therapy using fluorescent dye conjugated Apoptosis-targeting peptide-1 (ApoPep-1).

Methods Human hepatocarcinoma Hep3B was used to establish NIS expressing cell line with different promoter strength. Hep3B-CMV-NIS (HCN) and Hep3B-5mmTERT-NIS (H5TN) cells were established using retro-viral system. Iodide accumulation was observed by I-uptake assay. ApoPep-1 was conjugated with fluorescent dyes (AHR-648) and provided by BioActs (Seoul, Korea). For inducing apoptosis, 111 MBq I-131 was treated in the HCN and H5TN cells. After I-131 treatment for 24 hr, immunofluorescence staining was performed using 0.02 M AHR-648. The induction of apoptosis was examined by confocal microscopy and the fluorescent signals were also analyzed by the MetaMorph® Software.

Results The different levels of NIS expression between HCN and TEN were monitored by western blot analysis. I-uptake of HCN cells was significantly 1.3 fold higher than that of H5TN cells. In immunofluorescence staining, exposed histone H1 was detected on the surface of the I-131 treated cells and successful binding of AHR-648 was observed in HCN cells. However, weaker binding of AHR-648 was observed in H5TN cells. In MetaMorph analysis, the fluorescent signals of AHR-648 in I-131 treated HCN cells were significantly 2.4 fold higher than non-treated cells (P < 0.05). In H5TN cells, I-131-Induced apoptosis did not show significant difference.

Conclusions ApoPep-1 is a good tool for detection of apoptosis. In this study, we could evaluate the correlation between the I-uptake and apoptosis using AHR-648