The levels of Mcl and FLIP are

The levels of Mcl-1 and FLIP are homeostatically controlled by transcriptional and posttranslational processes. Accumulating evidence suggested that intracellular signaling molecules such as Akt are crucial to the transcription of Mcl-1 and FLIP [30], [31]. The constitutive activation of Akt is detected in most malignancies including glioma and that is associated with poor prognosis [44]. Inactivation and downregulation of Mcl-1 have been demonstrated in indomethacin-induced apoptosis [31]. Currently, there is little information regarding the relationship between indomethacin treatment and FLIP expression. Our biochemical and pharmacological results provided the evidence that the inactivation of Akt is correlated well with indomethacin-mediated Mcl-1 and FLIP downregulation at the transcriptional level and the inactivation of Akt contributes to the induction of apoptosis. Our results further showed that the inactivation of Akt downregulated the expression of both Mcl-1 and FLIP, promoting Bax mitochondrial distribution, elevating caspase activity and causing apoptotic cell death. Other reports indicate that Akt has additional effects on Mcl-1 levels in a process involving proteosomal degradation and phosphorylation [13]. Since Akt showed a positive effect in Mcl-1 and FLIP transcriptional control, we did not assess the potential involvement of alteration in protein stability. Our data showed that Akt signaling was inhibited by indomethacin, as manifested by a drop in the phosphorylation level. That is, kinases and phosphatases could be potential downstream effectors in indomethacin-treated glioma cells to block Akt activity. PP2A represents a crucial phosphatase in decreasing Akt phosphorylation [18,23,29]. Although cell-free study suggested an inhibitory effect of indomethacin on PP2A [20], a moderate PP2A activation was detected in indomethacin-treated glioma cells, indicating its potential involvement in indomethacin-induced Akt dephosphorylation. The biological relevance of PP2A activation in indomethacin-treated glioma cells was supported by the findings that its inhibition by okadaic AICAR phosphate attenuated indomethacin-induced viability loss, caspase 3 activation, Akt dephosphorylation, and Mcl-1/FLIP mRNA downregulation. In glioma cells, PP2A inhibition upregulates Akt activity and promotes cell proliferation [34]. Therefore, the upstream activators of PP2A likely show crucial contribution in converging signals from indomethacin to generate apoptosis. A growing body of evidence shows that ceramide is one upstream inhibitor of Akt through the activation of PP2A and ceramide has been implicated in the induction of apoptosis [14], [22], [25], [26], [36], [47]. Ceramide is produced either from de novo synthesis by ceramide synthase or from the hydrolysis of membrane sphingomyelin by neutral sphingomyelinase and acidic sphingomyelinase [18], [23], [25]. Clinically, the levels of ceramide are inversely correlated with the malignancy of glioma [37]. Ceramide induces glioma cell apoptosis involving Bcl-2 and FLIP downregulation and its production and proapoptotic action can be inhibited by Akt activation or Bcl-2 overexpression [17], [24], [39], [47]. W found that indomethacin increased intracellular ceramide levels in glioma cells. Biologically active C2-ceramide caused biochemical changes similar to those found in indomethacin-induced apoptotsis. Such changes included PP2A activation, Akt dephosphorylation, downregulation of both Mcl-1 and FLIP mRNA, and Bax mitochondrial distribution. Findings suggest that ceramide might be one candidate in mediating indomethacin-induced PP2A activation and glioma apoptosis. NSAIDs have a positive effect in sphingolipid biosynthesis and indomethacin can activate phospholpiase A2/arachidonic acid/sphingomyelinase pathway thereby elevating ceramide production [40], [41]. However, Backcross pathway is not likely a determining mechanism in our indomethacin-treatment. Neither phospholipase A2 inhibitors nor sphingomyelinase inhibitors showed a strong effect on indomethacin-induced Akt dephosphorylation and in glioma apoptosis (data not shown). Instead, ceramide synthase inhibitor showed moderate suppression on indomethacin-induced Akt dephosphorylation, caspase 3 activation, and glioma cell death. Although ceramide has been implicated in apoptosis, its contribution in NSAIDs-induced apoptosis in glioma was not elucidated. Our current findings in glioma suggested that ceramide production involving fumonisin B1-inhibitible ceramide synthase could be a switching molecule in indoemthacin-induced apoptosis to turn on the ceramide/PP2A/Akt axis.