we performed experiments to investigate whether selenite could influence the AKT survival pathway in CRC cells. We discovered that supranutritional doses of selenite time dependently inhibited the Src/PI3K/PDK1/AKT survival pathway in both SW480 CRC cells and HCT116, as shown in Figure 1a. In addition, in vitro PI3K and AKT assays showed that selenite Cediranib 288383-20-0 treatment inhibited AKT and PI3K activation in SW480 and HCT116 CRC cells. We consequently postulated that FoxO family proteins may be controlled by selenite inhibited AKT. To test this hypothesis, we immunoblotted FoxO family proteins in selenite treated samples and found that selenite constantly suppressed the phosphorylation of the proteins, indicating that FoxO proteins might be activated when AKT is restricted by selenite. To further corroborate this finding, we produced cytoplasmic Neuroblastoma and nuclear fractions from cells and immunoblotted for r Foxo3a and FoxO3a in both get a handle on and selenite treated samples and found that selenite increased the nuclear levels of FoxO3a but lowered its levels of phosphorylation. Moreover, immunofluorescence also supported the aforementioned that selenite induced FoxO3a accumulation in the nucleus. Taken together, these indicated that selenite inhibited Src/PI3K/PDK1/AKT signaling and activated FoxO family proteins in SW480 and HCT116 CRC cells. AKT/FoxO3a signaling is correlated with seleniteinduced apoptosis in CRC cells. Having found that selenite therapy inhibited Src/PI3K/PDK1/AKT signaling and activated FoxO proteins, we conducted a series of studies to investigate the relationship between FoxO3a and AKT in selenite induced apoptosis in CRC cells. On one hand, as revealed in Figures 2a and b, when AKT was inhibited in selenite handled CRC cells with either the PI3K inhibitor LY294002 or reversible HSP90 inhibitor AKT siRNA, we discovered that both treatments further reduced the p AKT level. Needlessly to say, inhibiting AKT more suppressed the phosphorylation of FoxO3a at Ser253 in spite of selenite therapy. Conversely, when we activated AKT in CRC cells using constitutively activated AKT constructs just before selenite treatment, we discovered that, in line with our theory, constitutively activated AKT enhanced phosphorylation of AKT and FoxO3a and selenite can no longer lower phosphorylation of AKT and therefore phosphorylation of FoxO3a. These jointly confirmed that seleniteelicited inhibition of AKT was from the activation of FoxO3a. Eventually, we attempted to determine the position of AKT/FoxO3a in selenite induced apoptosis of CRC cells. First, from western blot of the aforementioned examples, we observed that reactivation of AKT resulted in less cleavage of apoptosis related markers such as caspase 9 and PARP, although further inhibition of AKT led to additional cleavage of the apoptosis related markers.