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Transient adenoviral expression of AC compared with adenoviral expression of green fluorescent protein also revealed increased Akt phosphorylation in MIA, Panc01, SCC14A, PPC1 and DU145 cells, suggesting a generalizable phenomenon of AC induced Akt activation in cancer. Furthermore, shRNA delivered by adenovirus decreased pAkt. In order to validate that we are observing functional signaling Lenalidomide through Akt when we express AC, we probed for phosphoproteins downstream of Akt. We observed activation of the mammalian target of rapamycin pathway, as well as inhibition of GSK 3beta, which is involved in regulation of cell proliferation and metabolism. 16 SphK1 mediates AC induced Akt activation The bioactive lipids ceramide, sphingosine and S1P have all been linked to the regulation of Akt. We observed no change in total cell ceramide in Ad AC infected PPC1 cells Gene expression compared with Ad GFP, though species specific alterations were observed. Sphingosine and S1P were significantly elevated in Ad AC infected cells. In order to measure secreted S1P, we treated Ad AC/GFP infected PPC1 cells with C17 C6 ceramide, finding significant C17 S1P increase in the cells and medium. Treatment of cells with exogenous sphingosine did not activate Akt, rather decreasing pAkt moderately after 6 h of treatment. Addition of the dual isoform sphingosine kinase inhibitor SKI?II decreased Akt activation at 6 h, and did not augment Akt activation alone or in combination with sphingosine. We then infected PPC1 cells with Ad AC or Ad GFP in the presence of SKI?II, and observed a dose dependent reduction in Akt activation, suggesting that sphingosine kinase activity is necessary for AC induced Akt activation. Infection of wild type or sphingosine kinase 2 knocked out mouse embryonic fibroblasts with Ad AC promoted strong activation of Akt, Cediranib whereas AC had no impact on Akt activation in SphK1 KO MEFs. Ad AC increased S1P cell content and secretion into the medium in WT and SphK2 KO MEFs, but not in SphK1 KO MEFs. To confirm the observation that SphK1 may be necessary for AC induced Akt activation, we used shRNA and small interfering RNA to knock down each SphK isoform and confirmed that knockdown of SphK1, but not SphK2, abrogated AC induced Akt activation. S1PR2 stimulates PI3K to activate Akt To determine whether AC/S1P induced Akt activation was mediated by S1PRs, we expressed AC in PPC1 cells in the presence of the S1PR1 antagonist W146, or the S1PR2 antagonist JTE013. Whereas W146 had no impact on reducing AC induced Akt activation, JTE013 strongly inhibited AC induced Akt activation. W146 was validated in Supplementary Figure 3. Similarly, AC induced Akt activation was also prevented by JTE013 in WT MEFs, confirming that this phenomenon is intact in PTEN positive as well as PTEN negative cells. When we transfected PPC1 cells with shRNA sequences against S1PR1 S1PR2 or S1PR3, Ad AC induced Akt activation was unaffected in multiple S1PR1 and 3 knocked down cells, despite 60?70% reduction in mRNA.