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

为了明确细胞膜表面神经节苷脂GM3的含量与抗癌药物敏感性的关系，本研究以取自JCRB的14株人口腔鳞状细胞癌细胞（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）为研究对象。用薄层色谱法检测细胞中神经节苷脂的含量。Real - time PCR检测各细胞系GM3合成酶（SAT-1）基因的表达。不同细胞系GM3的表达量和SAT-1的表达量均有差异。根据MTT法得到的剂量-反应曲线计算顺铂的50%有效剂量（Ic50）。14株细胞系中，顺铂Ic50与SAT-1表达呈正相关（R=0.717）。接下来，在Ca9-22细胞系中控制GM3的表达后，检测顺铂敏感性。选择Ca9-22细胞系转染SAT-1基因，是因为其具有丰富的GM3前体LacCer和少量的GM3。与野生型相比，转染SAT-1的无性系GM3含量没有增加，但GM2含量有所增加。在转染SAT-1基因的克隆与野生型之间，顺铂敏感性未见差异。葡萄糖神经酰胺合成酶抑制剂D-PDMP、PBPP和P4检测神经节苷脂对Ca9-22、0SC-20和SCC-4细胞系的作用，对顺铂和依托泊苷的敏感性无明确影响。怀疑GM3单独与顺铂敏感性无关，可能有其他因素参与。神经节苷脂在抗癌药物诱导细胞凋亡的信号通路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.