耐药人乳腺癌细胞中葡萄糖神经酰胺合成酶的表达及意义

Expression of Glucosylceramide Synthase in Human Doxorubicin-resistant Breast Cancer Cells and Its Significance

目的:探讨葡萄糖神经酰胺合成酶(GCS)基因在人乳腺癌细胞株(MCF-7)和耐阿霉素人乳腺癌细胞株(MCF-7/ADR)中的表达及意义。方法:采用MTT法检测阿霉素对MCF-7/ADR和MCF-7的半数抑制浓度(IC50),用不同浓度的GCS抑制剂PDMP预处理MCF-7/ADR不同时间后检测IC50;运用流式细胞术检测MCF-7及PDMP预处理MCF-7/ADR前、后GCS蛋白的表达水平;应用实时荧光定量PCR法检测MCF-7及PDMP作用前、后MCF-7/ADR中GCS基因的表达(GCSN)水平。结果:阿霉素对MCF-7/ADR和MCF-7的IC50分别为(18.95±0.54)、(0.84±0.07)μg·mL-1;MCF-7/ADR对MCF-7的耐药倍数为22.69倍,PDMP作用后阿霉素对MCF-7/ADR的IC50下降至(5.63±0.58)μg·mL-1。MCF-7/ADR中GCS蛋白及GCSN的表达均高于MCF-7(P<0.01),PDMP使MCF-7/ADR中GCSN表达水平从0.0048±0.0017下降至0.0021±0.0004。结论:GCS可能参与肿瘤耐药的发生过程,并在MCF-7/ADR多药耐药中起重要作用。

OBJECTIVE： To investigate the expression of glucosylceramide synthase（GCS） gene in human MCF - 7 cells and MCF-7/ADR （doxorubicin-resistant） breast cancer cells and its significance. METHODS： MTT was used to detect the IC50 of MCF - 7 and MCF - 7/ADR to doxorubicin; exposing MCF - 7/ADR to various concentrations of PDMP for different time, then the IC50 of MCF - 7/ADR was detected. FCM was employed to determine the expression of GCS protein in MCF - 7 and MCF - 7/ADR before and afer pretreatment with PDMP ; real time RT - PCR was adopted to evaluate the expression of GCSN in them. RESULTS： The IC50 of MCF-7/ADR and MCF-7 were （18.95± 0.54） and （0.84±0.07）ug·mL^-1 the drug resistance of MCF- 7/ ADR was 22.69 times that of MCF- 7. PDMP exposure enhanced doxorubicin sensitivity, it decreased the IC50 of MCF - 7/ADR to （5.63±0.58） ug·mL^-1. The expressions of both GCS and GCSN in MCF - 7/ADR were significantly increased compared to MCF- 7 cells by FCM and real time RT- PCR （P （0.01） . PDMP exposure decreased of the expression of GCSN from 0.004 8± 0.001 7 to 0.002 1±0.000 4. CONCLUSIONS： GCS might be involved in the development of drug resistance in malignant carcinoma and play an important role in the drug resistance of MCF- 7/ADR.