## 4 Discussion
Thus, apparently only our in vivo data seem conflicting. The association of secstanibi uptake with both proliferation index and apoptosis could be considered an instrumental/clinical manifestation of pathophysiological mechanisms commonly involved in breast cancer development. However, a molecular characterization of the events associated with the accumulation of secstanibi into the mitochondria could open new clinical perspectives in the management of breast cancer cells. In fact, the regulation of the mitochondria related apoptotic process is currently one of the main targets of anti-cancer therapies. In this context, the preliminary data of [PERSON] and colleagues suggested that the secstanibi uptake correlated with apoptosis levels in breast cancer tissues following chemotherapy treatment [23]. In this study, the biological link between secstanibi uptake and the apoptotic phenomenon has been investigated in vitro by using BT-474 breast cancer cell lines. In particular, three different concentrations of [99 mTc]Tc-Sestamibi (10 \(\upmu\)g/mL, 1 \(\upmu\)g/mL, 0.1 \(\upmu\)g/mL) have been used to investigate the response of breast cancer cells to secstanibi uptake. Concerning the proliferation index, a significant decrease was observed in breast cancer cells incubated with a high concentration of [99 mTc]Tc-Sestamibi (10 \(\upmu\)g/mL). It is important to note that both in the control and at a lower concentration of [99 mTc]Tc-Sestamibi (0.1 \(\upmu\)g/mL), no decrease in proliferation index was observed after 120h. Excluding the experiment with a high concentration of [99 mTc]Tc-Sestamibi (10 \(\upmu\)g/mL), in vitro data confirmed the uptake of secstanibi in breast cancer cells during the proliferation phase. Amazingly, in vitro investigations showed a significant increase in caspase-3-positive cells in cultures incubated with 10 \(\upmu\)g/mL of 99 mTc-sestamibi. In particular, after 72 h, the presence of secstanibi in the cell medium induced a complete inversion between the proliferation index and apoptosis. In fact, at this time point, the number of caspase-3-positive cells was greater than those positive to the Ki67 markers. From a morphological point of view, these cell cultures showed numerous dead cells characterized by typical apoptotic sign such as pyknosis, or karyopyrknosis. In addition, transmission electron microscopy investigations allowed us to evaluate the ultrastructure of breast cancer cells incubated with [99 mTc]Tc-Sestamibi, demonstrating a progressive increase in the presence of apoptotic alterations. These damages were associated with both [99 mTc]Tc-Sestamibi concentration and incubation time. Remarkably, EDX investigations also showed the presence of [99 mTc]Technetium in the cytoplasm of apoptotic cells. Altogether, the data of this study suggest that, in the early phases, secstanibi uptake occurs mainly in breast cancer cells with a high proliferation index because of the high metabolic cell activity and the subsequent increase in the mitochondrial membrane potential. Then, the accumulation of secstanibi in the breast cancer cell mitochondria can induce an alteration of normal homeostasis, thus triggering the apoptotic event.