The key finding of our present study is represented by the identification of a new chain of kinase reaction in glioma cells, activated by upregulated SPHK1, sequentially causing phosphorylated activation of Akt, phosphorylated inactivation of FOXO3a and downregulation of Bim, and consequently leading to evasion of glioma cells from programmed cell death or abrogation of chemotherapeutic agents-induced apoptosis.
Evasion of apoptosis is a hallmark of most human tumors and constitutes an important clinical problem. Many therapeutic agents act against cancers through inducing apoptosis, and resistance to cancer therapies usually involves intrinsic or extrinsic abrogation of the apoptotic mechanism in cancer cells. Accumulating evidences have indicated that SPHK1 could protect cancer cells from apoptosis 
. Therefore, understanding the molecular mechanisms that mediate the anti-apoptotic effect of SPHK1 may lay a foundation for new anti-cancer strategies. To this end, the present study has for the first time provided a molecular dissection of an SPHK1-activated signal transduction cascade that leads to down-regulation of Bim and consequent resistance to apoptosis.
Bim, a member of Bcl-2 family, is a pro-apoptotic protein characterized by containing a single Bcl-2 homology 3 domain. In line with previous studies 
, the expression of Bim appears to be under the regulation of SPHK1in glioma cells. Furthermore, overexpression of SPHK1 correlated with downregulation of Bim in clinical glioma samples. Interestingly, induction of Bim expression has also been linked to apoptosis induced by chemotherapeutic agents such as lovastatin, anisomycin-caused ribotoxic stress, or nitric oxide 
. Thus, our finding provides a new insight into a possible mechanism that underlies SPHK1 induced resistance of glioma cells to apoptosis.
It is of note that regulation of Bim protein is attributed to transcriptional and posttranslational mechanisms 
. One of the transcription factors involved in modulation the expression of Bim is a forkhead transcription factor, FOXO3a. Consistent with previous findings 
, our study identifies FOXO3a as a mediator of SPHK1-associated downregulation of Bim, based on the following observations. Firstly, SPHK1 overexpression caused FOXO3a phosphorylation, and SPHK1 knockdown caused FOXO3a dephosphorylation in glioma cells. Secondly, pharmacological inhibitor of SPHK1 suppressed FOXO3a phosphorylation and increased Bim level. Thirdly, the transcriptional activity of FOXO3a was regulated by the level of SPHK1 protein in glioma cells.
It is evident that FOXO3a is under the regulation of the PI3K/Akt pathway 
. Akt seems to be essential in phosphorylating FOXO3a in current study because our results showed activation of Akt and Ser253
phosphorylation of FOXO3a in SPHK1-overexpressing glioma cells, as well as Akt inactivation and Ser253
dephosphorylation of FOXO3a in SPHK1-downregulated glioma cells. The results of our study using LY249002 to suppress PI3K further confirmed that activation of FOXO3a was attributable to deregulation of PI3K/Akt signaling. Thus, data obtained from the present investigation indicate that PI3K/Akt signaling may play a role in mediating the regulation of FOXO3a by SPHK1. Furthermore, studies have demonstrated that SPHK1 can activate the PI3K/Akt pathway in human cancers 
. In glioma cells, as shown by our study, upregulated SPHK1 activates PI3K/Akt signaling and supports cell survival through modulation of FOXO3a and Bim, representing a new mechanism possibly underlying the development of glioma and probably, its resistance to pro-apoptotic therapeutics. Nevertheless, mechanism by which SPHK1 activates PI3K in glioma cells, i.e., via a direct versus indirect interaction between two molecules, remains to be determined. In this context, it is of note that sphingosine, sphingosine 1-phosphate (S1P) and ceramide are inter-convertible sphingolipids associated with numerous cell functions including proliferation, apoptosis and migration 
. It has been demonstrated that Ceramide mediates and triggers cell growth arrest or apoptosis by inactivating PI3K/Akt 
, whereas S1P enhances proliferation and inhibits ceramide-mediated apoptosis 
. Furthermore, SPHK1 is a key regulator of the ceramide/S1P biostat since it is responsible for phosphorylating sphingosine, a catabolite of ceramide, to form S1P 
. One may postulate that SPHK1-induced PI3K/Akt activation is attributable to decreased levels of sphingosine and ceramide. Alternatively, another possibility can be linked to extracellular S1P signaling via the S1P1 G protein-coupled receptor, which activates Rac, Fak and PI3K 
. Further studies that aim at completely delineating the interaction between SPHK1 and PI3K/Akt will help develop new molecular targets for glioma therapy.
Treatment of glioma remains a clinical challenge. Currently available therapeutic strategies against glioma generally only delay local progression, and recurrence associated with resistance to therapies largely contributes to the high mortality of the disease. However, malignant glioma are typically infiltrative in nature. Thus, gaining new insights into the mediators of the apoptotic response in glioma will enable the development of novel anti-tumor strategies. In this context, targeting SPHK1 or its downstream signaling molecules as identified by our present study might represent new and potential strategies against human glioma.