we performed experiments to investigate whether selenite can

we conducted studies to research whether selenite can influence the AKT survival pathway in CRC cells. As shown in Figure 1a, we discovered that supranutritional doses of selenite time dependently inhibited the Src/PI3K/PDK1/AKT survival pathway in both HCT116 and SW480 CRC cells. Moreover, in vitro PI3K and AKT assays showed that selenite ATP-competitive ALK inhibitor treatment inhibited PI3K and AKT activation in HCT116 and SW480 CRC cells. We consequently postulated that FoxO family proteins could be governed by selenite restricted 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 could be triggered when AKT is inhibited by selenite. To further corroborate this finding, we produced cytoplasmic Inguinal canal and nuclear fragments from cells and immunoblotted for r Foxo3a and FoxO3a in both control and selenite treated samples and found that selenite increased the nuclear levels of FoxO3a but lowered its levels of phosphorylation. More over, immunofluorescence also supported the above mentioned 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 discovered that selenite treatment inhibited Src/PI3K/PDK1/AKT activated and signaling FoxO proteins, we conducted a series of experiments to analyze the relationship between AKT and FoxO3a in selenite induced apoptosis in CRC cells. On one hand, when AKT was inhibited in selenite addressed CRC cells with both the PI3K inhibitor LY294002 or Gemcitabine clinical trial AKT siRNA, as revealed in Figures 2a and b, we discovered that both treatments further lowered the p AKT level. Suppressing AKT further suppressed the phosphorylation of FoxO3a at Ser253 despite selenite therapy, needlessly to say. Conversely, when we activated AKT in CRC cells applying constitutively activated AKT constructs prior to selenite therapy, we found that, in line with our theory, constitutively activated AKT enhanced phosphorylation of AKT and FoxO3a and selenite could no further lower phosphorylation of AKT and therefore phosphorylation of FoxO3a. These collectively confirmed that seleniteelicited inhibition of AKT was from the activation of FoxO3a. Subsequently, we attempted to look for the function of AKT/FoxO3a in selenite induced apoptosis of CRC cells. First, from western blot of the above mentioned samples, we observed that reactivation of AKT triggered cleavage of apoptosis related markers such as PARP and caspase 9, although further inhibition of AKT led to additional cleavage of those apoptosis related markers.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>