Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation
Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation
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DOI：
10.1074/jbc.m900735200
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发表时间：
2009-04-17
影响因子：
4.8
通讯作者：
Banno, Yoshiko
中科院分区：
文献类型：
--
作者：
Nemoto, Satoshi;Nakamura, Mitsuhiro;Banno, Yoshiko
研究了人结肠癌细胞系中鞘氨醇激酶（SPHK）和细胞神经酰胺浓度与化疗敏感性的关系。在9种结肠癌细胞系中，SPHK 1和SPHK 2的活性和蛋白表达在RKO细胞中最高，在HCT 116细胞中最低。活力测定显示，HCT 116细胞对奥沙利铂（L-OHP）的作用敏感，而RKO细胞对L-OHP的作用耐药。用5 μ g/ml L-OHP处理诱导HCT 116细胞中各种神经酰胺（C16、C24、C24：1）的显著时间依赖性增加，但在RKO细胞中没有，如液相色谱/质谱所示。神经酰胺和半胱天冬酶的激活诱导的L-OHP在敏感的HCT 116细胞的增加被废除的预处理与中性鞘磷脂酶抑制剂，这表明神经酰胺的形成是由于中性，而不是酸性，鞘磷脂酶的激活。相反，在L-OHP抗性RKO细胞中，用SPHK抑制剂或通过RNA干扰沉默SPHK 1和SPHK 2的处理抑制了细胞活力，并在L-OHP处理后增加了半胱天冬酶活性和细胞神经酰胺形成。由SPHK抑制和L-OHP诱导的神经酰胺形成升高被伏马菌素B1抑制，但不被多球壳菌素抑制，表明神经酰胺形成是通过补救途径。内源性磷酸化Akt水平在抗性RKO细胞中比在敏感HCT 116细胞中高得多。RKO细胞中SPHK 1或SPHK 2沉默降低磷酸化Akt水平，增加p53和p21蛋白水平以及响应于L-OHP处理的聚（ADP-核糖）聚合酶切割。这些发现表明，SPHK亚型和中性鞘磷脂酶有助于通过控制神经酰胺的形成和下游Akt途径在人结肠癌细胞的化疗敏感性的调节。
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 5 mu g/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.