Therefore, the 2 S6K homologs had distinct effects on TNF- and TRAILinduced cell death. Since silencing of S6K1 caused a modest inhibition of TNF- and TRAIL-induced apoptosis , and S6K1 was shown to negatively regulate Akt via a feedback loop , we examined if knockdown of S6K1 enhances TNF-induced activation of Akt in MCF-7 cells. Figure 2 exhibits that depletion of S6K1 in MCF-7 breast cancer cells enhanced phosphorylation of Akt. In contrast to S6K1, knockdown of S6K2 decreased both basal and TNF-induced Akt phosphorylation . Depending on densitometric scanning of 4 independent experiments, knockdown of S6K2 decreased basal and TNF-induced Akt phosphorylation at Ser473 by 40% and 60%, respectively . We also examined the consequence of S6K2 knockdown on Akt phosphorylation in ZR-75-1 and MDA-MB-231 breast cancer cells . Knockdown of S6K2 decreased Akt phosphorylation, and enhanced PARP cleavage and caspase activation in ZR-75-1 cells . TNF had very little impact on cell death in MDA-MB-231 cells .
On the other hand, S6K2 depletion failed to boost cell death in response to TRAIL in MDA-MB-231 cells . In contrast to MCF-7 cells, which lack caspase-3, ZR-75-1 and MDA-MB-231 cells include practical caspase-3. Given that Akt is known as a substrate for caspase-3, apoptotic selleck chemicals tsa hdac stimuli can also induce cleavage of Akt and this may well contribute to decrease in Akt level in response to TNF or TRAIL. Because knockdown of S6K2 inhibits Akt phosphorylation, we examined if S6K2 promotes cell survival through Akt. We examined the capability of constitutively-active Akt to reverse the potentiation of cell death due to S6K2 depletion. Figure 4A exhibits the adenoviral vector-mediated delivery of CA-Akt in MCF-7 cells decreased TNF-induced PARP cleavage in contrast to cells transfected with adeno-GFP.
When knockdown of S6K2 brought about a substantial enhance in TNF-induced PARP cleavage, selleck chemicals buy Tandutinib overexpression of CA-Akt inhibited TNF-induced PARP cleavage in S6K2-depleted cells. Equivalent benefits have been obtained when we monitored cell death by staining cells with Annexin V and PI . These results propose that S6K2 mediates its prosurvival result by way of Akt. Though TNF and TRAIL set off cell death via the receptor-initiated pathway, they might also amplify cell death by way of the mitochondrial pathway . To determine the mechanism by which depletion of S6K2 potentiates TNF-induced cell death, we monitored TNF-induced caspase activation and processing of Bid. Figure 5A exhibits that TNF brought on an increase in phospho-Akt which was attenuated by S6K2 knockdown. Depletion of S6K2 was associated with enhanced processing of PARP and procaspase-8 in response to TNF.
This was accompanied by an increase inside the cleavage of Bid, a substrate for caspase-8 and enhanced processing of procaspase-9, the apical caspase from the mitochondrial cell death pathway. We also compared the results of S6K1 and S6K2 knockdown on cellular responses to TRAIL .