Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation

Abstract

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5μg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells. The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5μg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells. Sphingolipids have critical functions as signaling molecules. In particular, ceramide and sphingosine 1-phosphate (S1P) 3The abbreviations used are: S1P, sphingosine 1-phosphate; l-OHP, oxaliplatin (cis-(oxalato(1R,2R-cyclohexanediamine)platinum); SPHK, sphingosine kinase; SMase, sphingomyelinase; nSMase, neutral sphingomyelinase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PARP, poly(ADP-ribose) polymerase; SKI, sphingosine kinase inhibitor (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole); MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; ERK, extracellular signal-regulated kinase; mitogen-activated protein kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; siRNA, small interfering RNA; Z, benzyloxycarbonyl. are involved in regulating cell responses such as proliferation and apoptosis (1Taha T.A. Hannun Y.A. Obeid L.M. J. Biochem. Mol. Biol... 2006; 39: 113-131Google Scholar, 2Ogretmen B. Hannun Y.A. Nat. Rev. Cancer.. 2004; 4: 604-616Google Scholar, 3Olivera A. Kohama T. Edsall L.C. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol... 1999; 147: 545-558Google Scholar-4Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem... 1999; 274: 34499-34505Google Scholar). Ceramide, located at the central position of the sphingolipid metabolic pathway, is both the precursor and degradation product of sphingomyelin. Ceramide acts as a signaling molecule to activate a variety of signaling cascades that ultimately lead to cell responses such as apoptosis, growth arrest, cell differentiation, and various aspects of inflammation (5Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta.. 2002; 1585: 114-125Google Scholar). The hydrolysis of sphingomyelin is catalyzed by sphingomyelinases (SMases) yielding ceramide and phosphorylcholine. To date, several SMase activities have been identified that can be classified in two main groups, the acidic and neutral forms. Ceramide can also be formed from sphinganine by ceramide synthase. This de novo synthesis of ceramide requires hours of stress exposure and can be induced by several factors including tumor necrosis factor-α, hypoxia, and various chemotherapeutics (6Linn S.C. Kim H.S. Keane E.M. Andrai L.M. Wang E. Merrill Jr. A.H. Biochem. Soc. Trans... 2001; 29: 831-835Google Scholar). Ceramide is also produced by the salvage pathway. Ceramide is involved in the activation of both downstream and upstream caspases (7Takeda Y. Tashima M. Takahashi A. Uchiyama T. Okazaki T. J. Biol. Chem... 1999; 274: 10654-10660Google Scholar). Ceramide also triggers the activation of various protein kinase cascades, including the classical MAPK/ERK cascade (8Raines M.A. Kolesnick R.N. Golde D.W. J. Biol. Chem... 1993; 268: 14572-14575Google Scholar) and the more recently discovered stress-activated protein kinases of the SAPK/JNK subfamily (9Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature.. 1996; 380: 75-79Google Scholar). Another signaling pathway involved in the regulation of an apoptosis cell response that may be affected by ceramide is the survival phosphatidylinositol 3-kinase (PI3K)/Akt cascade (10Schubert K.M. Scheid M.P. Duronio V. J. Biol. Chem... 2003; 275: 13330-13335Google Scholar). On the other hand, the sphingolipid S1P acts as a potent mitogen for a variety of cell types (11Spiegel S. Milstein S. Nat. Rev. Mol. Cell Biol... 2003; 4: 397-407Google Scholar, 12Milstien S. Spiegel S. Cancer Cell.. 2006; 9: 148-150Google Scholar), mainly involving specific cell surface S1P receptors (13Hla T. Pharmacol. Res... 2003; 47: 401-407Google Scholar) that belong to the class of G protein-coupled receptors (S1P(1,2,3,4,5)) (14Young N. van Brocklyn J.R. Scientific World Journal.. 2006; 6: 946-966Google Scholar). S1P prevents apoptosis induced by serum withdrawal, Fas, and ceramide, among others. A critical factor regulating the availability of S1P is the activity of sphingosine kinase (SPHK), sphingosine to S1P and is to as an in S. Spiegel S. Cancer Cell.. 2006; 9: 148-150Google Scholar, T. Pharmacol. Res... 2003; 47: 401-407Google Scholar, S. Spiegel S. Biochim. Biophys. Acta.. 2006; Scholar). SPHK have been SPHK1 and SPHK1 activity is by various growth factors and S. Spiegel S. Biochim. Biophys. Acta.. 2006; Scholar), whereas the activation of SPHK2 is growth factor SPHK2 activity in the cancer cell S. A. M. S. Spiegel S. J. Biol. Chem... Scholar). of SPHK1 proliferation and cells from apoptosis, and tumor formation in (11Spiegel S. Milstein S. Nat. Rev. Mol. Cell Biol... 2003; 4: 397-407Google Scholar, 12Milstien S. Spiegel S. Cancer Cell.. 2006; 9: 148-150Google Scholar, T. B.J. J. T. M.J. Hannun Y.A. Obeid L.M. 2006; Scholar). On the other hand, SPHK2 been to growth and apoptosis S. M. S. Spiegel S. J. Biol. Chem... 2003; Scholar). the of SPHK2 not been as of SPHK2 in cancer cells in cell growth inhibition Brocklyn J.R. J. Scholar). These two ceramide and S1P, a cellular regulating the between cell growth and cell SPHK1 is in a variety of T. B.J. J. T. M.J. Hannun Y.A. Obeid L.M. 2006; Scholar, Y. Cancer Res... 2003; Scholar, M. E. N. V. P. J. B. Cuvillier O. Cancer Res... Scholar), and ceramide levels are in colon with M. A. M.A. Hannun Y.A. Cancer Res... 2001; Scholar). the in of ceramide may have effects in cancer of cellular the between ceramide and S1P to be a critical of cell and proliferation and may be an factor to in tumor S. Spiegel S. Cancer Cell.. 2006; 9: 148-150Google Scholar, M. E. N. V. P. J. B. Cuvillier O. Cancer Res... Scholar, Y. A. C.E. J. Obeid L.M. B. J. Biol. Chem... Scholar). have that cancer cells with SPHK1 activity are resistant to the Y. Y. Y. N. Kim T. Y. Y. Biochem. Biophys. 2006; Scholar), and cells with SPHK1 activity are resistant to S. T. A. R. M. A. T. Y. Y. Y. R. M. A. T. T. 2006; Scholar, S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). In those the relationship between ceramide levels and SPHK activity was not In the ceramide levels and SPHK activity in and colon cancer cells. colon cancer cell various levels of SPHK1 and SPHK2 cancer cells with SPHK1 and SPHK2 expression were resistant to oxaliplatin with those with SPHK activities the increase in ceramide, and inhibition of both SPHK increased ceramide the colon cancer cells sensitive to l-OHP. The regulation of to by the sphingolipid through Akt activation and p53 expression in the two types of colon cancer cells is myriocin, and fumonisin B1 were from poly(ADP-ribose) polymerase and were from Cell and were from SPHK1 and SPHK2 were as M. T. Y. Y. Y. Biochim. Biophys. Acta.. Scholar). was from a sphingosine kinase inhibitor and a neutral sphingomyelinase inhibitor, were from The assay was from and were from Cell and Cell colon cancer cell from were in cell viability cells were at a of in Cell viability of and cells was for by to the of and of human and were from as S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). was from for the are as and and and and and for are and or of and and were to the tumor cells in and expression were by and and by was to the S. T. A. R. M. A. T. Y. Y. Y. R. M. A. T. T. 2006; Scholar). The was as the of the by the were by in and The cell of was to and to The were serum of and was by with specific was used as the were the was with a activity of the two SPHK SPHK1 and was as S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). SPHK activity was in the of or in SPHK1 activity was in the of sphingosine and in SPHK2 S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). SPHK2 activity was with sphingosine as a with serum and in the of in SPHK2 activity is and SPHK1 activity is S1P was and by with as the The of S1P the was by and a The activity was as and neutral SMase activities were as (7Takeda Y. Tashima M. Takahashi A. Uchiyama T. Okazaki T. J. Biol. Chem... 1999; 274: 10654-10660Google Scholar). and activities were an assay were to the the cells were at in and the was with with or and for treatment with l-OHP. for the indicated the were to the by of with a and of S1P and cancer cells were by in the were a and acidic with and used as were the The were in and to were a liquid to a mass as S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). for were were for by sphingolipid by the of the or in sphingolipid was a concentration a for was in the Ceramide synthesis of ceramide from sphingosine was by sphingosine as S. J. V. Obeid M. in Scholar). of sphingosine was and in the The cell and were in the and for at The was by the the of and The were and to as J. Hannun Y.A. A. 2006; 39: Scholar). and were used to the The was by or of and A of than was between and of Cancer is an and the other SPHK been to be S. A. M. S. Spiegel S. J. Biol. Chem... Scholar). SPHK1 and SPHK2 activities were in nine human colon cancer cell lines. activities among the nine colon cancer cell Among cell lines, RKO cells the highest SPHK1 and SPHK2 whereas HCT116 cells the lowest and cells the activities SPHK1 and SPHK2 activities in RKO cells were and higher than those in HCT116 and cells, protein expression levels were higher in RKO cells than in HCT116 and cells, to activity levels to an l-OHP, in the cell lines. HCT116 cells were the sensitive to l-OHP was and RKO cells were the sensitive to l-OHP cells These that the activities and protein of SPHK isoforms may be to chemosensitivity for l-OHP. RKO cells with higher SPHK activity and expression were much sensitive to the with HCT116 cells with SPHK activity and were sensitive to the These to SPHK activity chemosensitivity in cells. in of after l-OHP in and levels have been involved in apoptosis of various cancer cells T. M. M. A. T. T. T. S. J. Biol. Chem... 2002; Scholar, S. C.E. S. J. T.A. Hannun Y.A. Obeid L.M. B. J. Biol. Chem... 2004; Y. J. Hannun Y.A. J. Biol. Chem... Scholar). To ceramide is involved in the of to l-OHP, was used to ceramide levels in and RKO cells, with or l-OHP treatment. Endogenous ceramide levels and ceramide were not among cell lines, and and levels of and but of in HCT116 cells with l-OHP revealed that the levels of both and and increased and and after a increase in or were not On the other hand, treatment of RKO cells with l-OHP not in of the ceramide cell was at l-OHP concentration in RKO cells. The treatment of cells with l-OHP for induced in the levels of and were than those of HCT116 cells These that the in ceramide levels are with l-OHP To the in ceramide levels are due to hydrolysis of and neutral SMase activities in both HCT116 and RKO cell after various of l-OHP treatment. SMase activity not l-OHP treatment in cell In contrast, neutral SMase activity was increased at in the HCT116 cells but not in the resistant RKO cells. These findings that the increased ceramide was due to neutral hydrolysis of sphingomyelin. of Ceramide by and in HCT116 HCT116 cells with a neutral SMase inhibitor, the of and activity increased in response to l-OHP for but was abolished by pretreatment of the cells with the caspase inhibitor The l-OHP increase in activity was suppressed by pretreatment but not by the de novo ceramide inhibitor fumonisin l-OHP treatment of HCT116 cells induced was suppressed by and with the of activity B1 not The of HCT116 cells was by treatment with and but not fumonisin B1 These that ceramide from sphingomyelin hydrolysis induced by neutral SMase was involved in the apoptosis of HCT116 cells. of of SPHK l-OHP of RKO and in RKO of the chemosensitivity Y. Cancer Res... 2003; Scholar, M. E. N. V. P. J. B. Cuvillier O. Cancer Res... Scholar), and SPHK1 activity with ceramide levels (1Taha T.A. Hannun Y.A. Obeid L.M. J. Biochem. Mol. Biol... 2006; 39: 113-131Google Scholar, 3Olivera A. Kohama T. Edsall L.C. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol... 1999; 147: 545-558Google Scholar). used an SPHK inhibitor, SKI, to SPHK activity l-OHP in the resistant RKO cells. the of for SPHK1 and SPHK2 Treatment of RKO cells with inhibited both SPHK1 and SPHK2 activity by a of for the two SPHK the of was ceramide in RKO cells. RKO cells were with SKI, l-OHP treatment induced in the levels of but not the treatment with and l-OHP degradation in RKO cells ceramide formation and cell was increased in the of in RKO cells These indicated that inhibition of SPHK activity the ceramide and l-OHP in the resistant RKO cells. SPHK inhibition was to increase ceramide RKO cells were with or the of involving de novo ceramide for and fumonisin B1 for ceramide synthase. of by the treatment with and l-OHP was abolished by pretreatment with fumonisin B1 but not On the other hand, fumonisin B1 pretreatment induced of sphingosine in the cells with and l-OHP. ceramide activity was by sphingosine as the the treatment of RKO cells with and l-OHP a increase in to the of sphingomyelin hydrolysis the ceramide in RKO cells with and l-OHP, was The of RKO cells with of expression but not These indicated that the ceramide formation induced by treatment with and l-OHP in RKO cells was due to the increase in ceramide the of ceramide in in RKO cells. cleavage by and l-OHP was by pretreatment with fumonisin B1 but not In fumonisin B1 suppressed the by the viability assay in activity were well with ceramide induced by l-OHP. In HCT116 cells, activity was by treatment with l-OHP and suppressed in the of the neutral SMase inhibitor In contrast, l-OHP not increase activity in RKO cells, the treatment with and l-OHP caspase activity in RKO cells. These that have a in activation and of of Akt in Cancer activation of Akt is involved in survival signaling in various cell types Rev. 1999; Scholar). To the of Akt in l-OHP Akt was in both sensitive HCT116 and resistant RKO cells. In the cells, the Akt levels were much higher in RKO cells than in HCT116 cells, Akt levels were Akt was to in HCT116 cells with l-OHP of HCT116 cells with but not fumonisin the cells from the in Akt On the other hand, Akt was not to be affected by treatment with l-OHP in RKO cells The Akt in RKO cells with was suppressed by l-OHP treatment. not Akt These in Akt levels by l-OHP were to be with those in activation and in the other by l-OHP treatment were also levels of kinase ERK, and were not between the two cell and also were not after l-OHP treatment not l-OHP treatment of HCT116 cells induced in the expression of and in a time-dependent with RKO cells in of of Ceramide Akt and in RKO the of SPHK isoforms in cellular used two of of SPHK1 and In RKO cells with two and two protein were of SPHK1 by two specific but not siRNA, cleavage and of SPHK2 by two specific was to increase cleavage to a or than for SPHK1 Either SPHK1 or SPHK2 increased by l-OHP treatment These for the in the inhibition of SPHK activity by the increased of and Akt by l-OHP was in both SPHK1 and SPHK2 RKO cells p53 and p21 protein were in both SPHK1 and SPHK2 cells. of increased and of protein expression induced by l-OHP were in RKO cells with other of SPHK1 or SPHK2 not These findings that both SPHK1 and SPHK2 are involved in the regulation of ceramide and chemosensitivity in colon cancer of SPHK1 and SPHK2 ceramide Akt and protein expression in RKO cells. RKO cells were with of of or with to the the cells were with or l-OHP for The were from the cells, and the cellular ceramide concentration was by as are as increase of cells with l-OHP as of RKO cells with of or were with or l-OHP the cell were to the and was used as the that the human cancer cell SPHK1 is resistant to Y. Y. Y. N. Kim T. Y. Y. Biochem. Biophys. 2006; Scholar) and also that the SPHK1 in from with and is well with to S. T. A. R. M. A. T. Y. Y. Y. R. M. A. T. T. 2006; Scholar). In the have that SPHK activity and protein expression are to l-OHP in colon cancer cell that are in SPHK expression and of l-OHP-resistant RKO cells with SPHK inhibitor or of SPHK1 and SPHK2 induced a marked increase in l-OHP that an increased SPHK expression was in the resistant cell from the cells, chemosensitivity and SPHK levels not These lead to that as well is involved in in colon cancer cells. The ceramide is to cell differentiation, and apoptosis B. Hannun Y.A. Nat. Rev. Cancer.. 2004; 4: 604-616Google Scholar, B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta.. 2002; 1585: 114-125Google Scholar), and that increase the ceramide levels in cancer cells lead to the of cell In the that in sensitive HCT116 cells, l-OHP treatment induced an increase in the cellular ceramide in cell and treatment with a neutral SMase inhibitor suppressed the increase in ceramide to the of cell In contrast, in l-OHP-resistant RKO cells, l-OHP treatment not the cellular ceramide levels or cell treatment with and l-OHP induced cell and was by a increase in ceramide These that an increase in cellular ceramide levels is for the of cell by the l-OHP and that cellular ceramide levels are by SPHK activity in colon cancer cells. Among the ceramide an increase in but not in or cell in cells T. M. M. A. T. T. T. S. J. Biol. Chem... 2002; Scholar). also that the was to chemosensitivity in cells S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). In contrast, other that an increase in or is for apoptosis S. C.E. S. J. T.A. Hannun Y.A. Obeid L.M. B. J. Biol. Chem... 2004; Scholar, Y. J. Hannun Y.A. J. Biol. Chem... Scholar). in the levels are to neutral SMase whereas in the or levels are to the de novo ceramide HCT116 cells indicated that l-OHP treatment a increase in and whereas levels were These in the ceramide were by treatment with the neutral SMase inhibitor but not by fumonisin that of ceramides from sphingomyelin hydrolysis rather than de novo On the other hand, in the l-OHP-resistant RKO cells, was of of the ceramide by l-OHP treatment This be due to the SPHK in inhibition of de novo ceramide A. T. A. T. J. Biol. Chem... Scholar, J. A. M. A.H. S. Mol. Cancer Res... Scholar). levels were increased by l-OHP treatment in RKO cells with or with SPHK1 or SPHK2 The ceramide induced by inhibition of the SPHK activity or SPHK be the salvage pathway, fumonisin but not myriocin, inhibited ceramide and increased sphingosine in RKO cells In the silencing of the ceramide These that the ceramide induced by or SPHK1 and SPHK2 is to increased ceramide activity and a sphingosine salvage pathway J. J. Hannun Y.A. J. Biol. Chem... Scholar). at have for the of ceramide in apoptosis, in the of ceramide by may the cell The sphingolipid is as the of cellular cells, Y. A. C.E. J. Obeid L.M. B. J. Biol. Chem... Scholar) that the of is with increased SPHK1 expression and S1P in in the also have that the of the ceramide is to the activation of and sphingomyelin in cells Y. M. Y. S. S. Y. Okazaki T. Cancer Res... 2004; Scholar). that expression of but not sphingomyelin the ceramide and is with to S. T. A. R. M. A. T. Y. Y. Y. R. M. A. T. T. 2006; Scholar, S. S. M. A. S. A. A. T. M. Y. M. Y. Y. T. J. Scholar). with the decreased ceramide levels induced by of SPHK1 activity with ceramide The findings that l-OHP and treatment of RKO cells, SPHK1 and SPHK2 increased ceramide levels and induced These that SPHK is with due to a in ceramide isoforms of SPHK, SPHK1 and have been and SPHK1 is critical for cell and cancer cells SPHK1 are more resistant to apoptosis induced by (1Taha T.A. Hannun Y.A. Obeid L.M. J. Biochem. Mol. Biol... 2006; 39: 113-131Google Scholar, S. Spiegel S. Biochim. Biophys. Acta.. 2006; Scholar). In contrast, much is the of cell growth and apoptosis, suggesting that SPHK1 and SPHK2 have in the S. M. S. Spiegel S. J. Biol. Chem... 2003; Scholar). SPHK1 been to be with SPHK2 is to be involved in in human cells S. T. A. R. M. A. T. Y. Y. Y. R. M. A. T. T. 2006; Scholar). SPHK2 cancer cells to apoptosis, suggesting that SPHK2 the between and apoptosis S. P. S. Spiegel S. Cancer Res... Scholar). in the the inhibition of SPHK1 or SPHK2 activity an with l-OHP to apoptosis in l-OHP-resistant RKO cells that of SPHK1 and SPHK2 the of l-OHP, as in decreased Akt and increased p53 expression and cleavage in RKO cells These that l-OHP is by both SPHK1 and SPHK2 in colon cancer cells. Ceramide such as and (5Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta.. 2002; 1585: 114-125Google Scholar, K.M. Scheid M.P. Duronio V. J. Biol. Chem... 2003; 275: 13330-13335Google Scholar), and such as and phosphatidylinositol 3-kinase (11Spiegel S. Milstein S. Nat. Rev. Mol. Cell Biol... 2003; 4: 397-407Google Scholar). p53 is a well tumor and in the p53 are the in human tumor M. B. Scholar). A of in have that cancer cells are resistant to l-OHP and S. L. N. P. Cancer 2003; Scholar, R. E. P. S. Cancer Res... 2002; Scholar, J. S. P. A. Cancer Res... 2004; Scholar, J. B. E. Cancer Res... M.J. M. J.M. J. Cancer.. 2004; Scholar). In various and in HCT116 cells and l-OHP-resistant RKO cells. in and expression were induced by l-OHP treatment in HCT116 cells, whereas expression of was in RKO cells suggesting that have in apoptosis induced by l-OHP in colon cancer cells. In have that the pathway is to mainly through from cellular apoptosis E. J. P. M. Cancer 2004; Scholar). This signaling cell survival and Akt cells from apoptosis by activation and the of tumor cells to A. T. A. T. J. Biol. Chem... Scholar, E. J. P. M. Cancer 2004; Scholar). Akt is in human cancer cells cancer and Akt a in the to in These of Akt are with that Akt is in l-OHP-resistant RKO cells with HCT116 cells, suggesting that the Akt activation pathway is with l-OHP have in that Akt activation through signaling is with l-OHP in colon cancer cells. SPHK been as a regulating factor for the sphingolipid that the of and through the of ceramide and sphingosine to S1P S. Spiegel S. Cancer Cell.. 2006; 9: 148-150Google Scholar). ceramide growth and apoptosis by Akt (10Schubert K.M. Scheid M.P. Duronio V. J. Biol. Chem... 2003; 275: 13330-13335Google Scholar, A. T. A. T. J. Biol. Chem... Scholar, S. M. P. S. E. T. A. Cancer Cell.. Scholar). In contrast, SPHK activation Akt may or may not be by S1P receptors Y. Y. M. T. Y. S. J. 2001; Scholar, J. J.M. J. Biol. Chem... Scholar). In RKO cells were with inhibition of Akt was not from that the activation of SPHK1 and SPHK2 ceramide Akt activation that inhibition or of SPHK1 and SPHK2 the of to of Akt activation by l-OHP treatment and have Akt in the of p53 is phosphorylated by Akt at in activation J. R. Z. A. J. Biol. Chem... 2004; Scholar). This in in a by J. B. E. Cancer Res... Scholar), that the induced expression of p53 in RKO cells is at the after treatment with whereas p53 levels not by exposure to the also the activation of the Akt pathway as a of cellular survival and in colon cancer cells, that inhibition of the Akt pathway by an SPHK inhibitor or SPHK is a to l-OHP the of l-OHP is a of regulation of Akt that colon cancer cell l-OHP is SPHK1 and SPHK2 ceramide formation and Akt in for the of an SPHK inhibitor and l-OHP be SPHK activity in colon may be a for and for with