¹¹¹In-DOTA-DPA-Zn(II) binding with increasing of phosphatidylserine expression in cells

Objectives A Zn (II)-dipicolylamine analog (DPA) conjugated with a fluorophore has been shown to target cells expressing phosphatidylserine (PS). In this study, DPA analogs with and without coordinated zinc were conjugated with NHS-DOTA through an available amine, radiolabeled with ¹¹¹In and evaluated for accumulation in cultured human breast cancer BT-474 cells expressing different levels of apoptotic PS.

Methods Using an Annexin V apoptosis detection kit and FACS analysis, up-regulation of apoptosis was evaluated in cells treated with suberoylanilide hydroxamic acid (SAHA) (0-10 µM) for 48 h. The ¹¹¹In labeled DPA analogs were incubated at 2.5 µg/ml with SAHA treated cells for 60 min and the cellular accumulations of radioactivity were measured by counting the lysed cell suspensions.

Results Treatment with SAHA upregulated PS expression levels of BT-474 cells and increased apoptosis from 22 % to 33 % of cells in a dosage-dependent manner. The accumulations of ¹¹¹In-DOTA-DPA-Zn(II) was significantly (p<0.05) higher than ¹¹¹In-DOTA-DPA in cells treated with SAHA at all concentration from 1 µM to 10 µM. Furthermore, while the accumulation of ¹¹¹In-DOTA-DPA-Zn(II) increased with increasing percent of apoptotic cells, the accumulation of ¹¹¹In-DOTA-DPA was constant. The increase in accumulation of ¹¹¹In-DOTA-DPA-Zn(II) was significantly (p<0.05) higher in cells treated with SAHA at 5 µM compared with 1 µM but not between 5 and 10 µM and therefore consistent with the results of SAHA-induced apoptosis.

Conclusions These preliminary results show that the increased cell binding of ¹¹¹In-DOTA-DPA-Zn(II) correlates with increasing PS expression and suggest that radiolabeled DPA coordinated zinc analogs may be promising candidates for apoptosis imaging agents