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本研究旨在探讨葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)抑制剂D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol(PDMP)hydrochloride对K562/A02细胞株柔红霉素(daunorubicin,DNR)耐药的逆转作用及其逆转机制。采用四甲基偶氮唑蓝(MTT)法检测不同浓度PDMP对K562细胞和K562/A02细胞的增殖抑制效应及其对K562/A02细胞DNR耐药逆转作用;用流式细胞术检测PDMP对K562/A02细胞内DNR浓度及细胞凋亡率的影响;半定量RT-PCR和Western blot方法检测K562及K562/A02细胞mdr1、GCS基因的表达以及PDMP对K562/A02细胞两种基因表达的影响。结果表明:DNR对K562和K562/A02细胞的IC50分别为0.23±0.02和7.15±0.24μg/ml,当PDMP≤20μmol/L时,对K562及K562/A02细胞株无明显生长抑制作用;20μmol/L和10μmol/L PDMP均能增加K562/A02细胞对DNR的敏感性,其耐药逆转倍数分别为2.59和1.69;两种浓度PDMP作用于耐药株48小时后能增加细胞内DNR浓度(p<0.05)和细胞凋亡率(p<0.01)。20μmol/L PDMP处理K562/A02细胞48小时后可在mRNA和蛋白水平下调GCS和mdr1基因的表达(p<0.01)。结论:PDMP可以增强K562/A02细胞对DNR的敏感性,其机制可能与增加细胞内药物浓度及细胞凋亡率、下调GCS和mdr1基因表达有关。
The purpose of this study was to investigate the effects of glucose, glucosylceramide synthase (GCS) inhibitor D, L-threo-1-phenyl-2-decanoylamino-3-morpholino- Reversal effect of drug resistance of daunorubicin (DNR) and its reversal mechanism. The inhibitory effect of different concentrations of PDMP on the proliferation of K562 and K562 / A02 cells and the reversal of drug resistance to DNR on K562 / A02 cells were detected by MTT assay. Flow cytometry was used to detect the effect of PDMP on K562 / / A02 intracellular DNR concentration and apoptosis rate; semi-quantitative RT-PCR and Western blot were used to detect the expression of mdr1 and GCS genes in K562 and K562 / A02 cells and the effect of PDMP on K562 / A02 cells. The results showed that the IC50 of DNR on K562 and K562 / A02 cells were 0.23 ± 0.02 and 7.15 ± 0.24μg / ml, respectively. No significant inhibition on the proliferation of K562 and K562 / A02 cells was observed when PDMP was less than 20μmol / L and 10μmol / L PDMP could increase the sensitivity of K562 / A02 cells to DNR, and their reversal rates were 2.59 and 1.69, respectively. PDMP at two concentrations for 48 hours increased the intracellular DNR concentration (p <0.05) and the rate of apoptosis (p <0.01). After treated with 20μmol / L PDMP for 48 hours, the expressions of GCS and mdr1 gene were downregulated at mRNA and protein levels (p <0.01). CONCLUSION: PDMP can enhance the sensitivity of K562 / A02 cells to DNR. The mechanism may be related to increasing intracellular drug concentration and apoptosis rate and down-regulating gene expression of GCS and mdr1.