A research of regulation of glycosphingolipids gene expression and anticancer drug sensitivity in human oral cancer cell lines

为了阐明细胞膜表面神经节苷脂GM 3的量与抗癌药物敏感性之间的关系，本研究对从JCRB获得的14株人口腔鳞状细胞癌细胞系（Ca 9 -22、HSC-2、HSC-3、HSC-4、Ho-1-N-1、Ho-1-U-1、KON、KOSC 2-c13、SAT、SAS、SCC-4、SKN-3、OSC-19、OSC-20）进行了研究。采用薄层层析法检测细胞中神经节苷脂的含量。通过真实的时间PCR测定每个细胞系的GM 3合成酶（SAT-1）基因的表达。对于GM 3的量和SAT-1表达，细胞系观察到定量差异。根据MTT法得出的剂量-反应曲线计算顺铂的50%有效剂量（Ic 50）。在14株细胞中，顺铂的IC 50与SAT-1的表达呈正相关（R=0.717）。接下来，在控制GM 3在细胞系Ca 9 -22中的表达后，检查顺铂敏感性。本研究选择了富含GM 3前体LacCer、GM 3含量低的Ca 9 -22细胞系作为SAT-1基因的转染细胞。然而，SAT-1转染的克隆没有比野生型增加GM 3，但是由GM 3组成的GM 2增加了。SAT-1基因转染的克隆与野生型之间的顺铂敏感性没有差异。用葡萄糖神经酰胺合成酶抑制剂D-P13、PBPP和P4检测神经节苷脂对Ca 9 -22、0 SC-20和SCC-4细胞株的作用，结果显示对顺铂和足叶乙甙的敏感性无明显影响。推测GM 3单独与顺铂敏感性无关，可能有更多因素参与。神经节苷脂在抗癌药物诱导细胞凋亡的信号转导中的作用机制有待进一步研究。

To clarify the relation between the amount of ganglioside GM3 on the surface of the cell membrane and the anti-cancer drug sensitivity, this research was done with 14 human oral squamous cell carcinoma cell lines which were obtained from JCRB (Ca9-22,HSC-2,HSC-3,HSC-4,Ho-1-N-1,Ho-1-U-1,KON, KOSC2-c13,SAT, SAS, SCC-4,SKN-3,OSC-19,OSC-20). The ganglioside contents were detected by thin layer chromatography for the cell lines. The expression of GM3 synthesis enzyme (SAT-1) gene of each cell line was measured by the Real time PCR. A quantitative difference was seen by the cell lines as for the amount of GM3 and the SAT-1 expression. The 50% effective doses (Ic50) of cisplatin were calculated from the dose-response curves which were derived from MTT assay. The Ic50 of the cisplatin showed positive correlation with SAT-1 expression (R=0.717) in 14 cell lines. Next, the cisplatin sensitivity was examined after controlling the expression of GM3 in the cell line Ca9-22. The Ca9-22 cell line was selected to transfect SAT-1 gene, because it has rich LacCer which is the precursor of GM3 and little amount of GM3. However, the SAT-1 transfected clone did not increase GM3 than a wild type, but GM2 which was made of GM3 had increased. The difference of the cisplatin sensitivity was not seen between the SAT-1 gene transfected clone and a wild type. Inhibitors of glucosylceramide synthase, such as D-PDMP, PBPP and P4 were used to detect the role of ganglioside on Ca9-22, 0SC-20 and SCC-4 cell lines, which showed no definite effect on sensitivity of cisplatin and etoposide. It was suspected that GM3 alone do not have a relation with the cisplatin sensitivity, and more factors may take part in. Further investigation should be done to detect the mechanism of ganglioside on the cell signaling which induce apoptosis by anti-cancer drug.