Sphingosine kinase isoforms regulate oxaliplatin sensitivity of human colon cancer cells through ceramide accumulation and Akt activation.
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 5microg/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.
Nemoto
Satoshi
S
Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
Nakamura
Mitsuhiro
M
Osawa
Yosuke
Y
Kono
Saki
S
Itoh
Yoshinori
Y
Okano
Yukio
Y
Murate
Takashi
T
Hara
Akira
A
Ueda
Hiroshi
H
Nozawa
Yoshinori
Y
Banno
Yoshiko
Y
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000187:drug effects*; D002518:Ceramides / Q000378:metabolism*; D024161:Collagen Type XI / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D000077150:Oxaliplatin; D010088:Oxidoreductases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000099157:Sphingosine Kinase
D000970:Antineoplastic Agents; D002518:Ceramides; D024161:Collagen Type XI; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D010088:Oxidoreductases; D017853:Phosphotransferases (Alcohol Group Acceptor); D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D013108:Sphingomyelin Phosphodiesterase; D000099157:Sphingosine Kinase; C499319:COL11A2 protein, human; C108138:dihydroceramide desaturase; C491484:SMPD3 protein, human