Wang H, Wang X, Hu R, Yang W, Liao A, Zhao C, Zhang J, Liu Z
Department of Hematology, Shengjing Hospital affiliated to China Medical University, Shenyang, China.
Department of Urology, Shengjing Hospital affiliated to China Medical University, Shenyang, China.
Cancer Gene Ther. 2014 Feb;21(2):83-9. doi: 10.1038/cgt.2013.87. Epub 2014 Jan 17.
Methylation of secreted frizzle-related protein (SFRP) genes activates Wnt/ß-catenin signaling and promotes tumor development. This study investigated whether SFRP5 gene methylation causes multidrug resistance (MDR) in leukemia through the Wnt/ß-catenin signaling, leading to the upregulation of the mdr1 gene and its product, P-glycoprotein (P-gp). Methylation-specific PCR identified SFRP5 gene methylation in cultured bone mononuclear cells from 7/12 patients with acute leukemia and in four human leukemia cell lines (HL-60, Raji, U937 and KG1a). Western blotting revealed absent SFRP5 protein expression in cells from 5/7 patients with SFRP5 gene methylation and in all cell lines. Treatment with a demethylation agent (DAC) rescued SFRP5 expression. mdr1 mRNA and P-gp protein were detected in cells from 3/5 patients with absent SFRP5, and in the KG1a cell line; these cells also had the highest levels of activated ß-catenin. In cells from these three patients, DAC rescued SFRP5 expression and downregulated mdr1 and P-gp. SFRP5 protein expression was rescued in transgenic KG1a/SFRP5 cells, compared with KG1a/eGFP or untransfected KG1a cells. mdr1 and P-gp in KG1a/SFRP5 cells were downregulated. Doxorubicin IC50 values were significantly lower in KG1a/SFRP5 (0.573±0.131 μM) than in KG1a (0.963±0.115) or KG1a/eGFP (0.917±0.138) cells (P<0.05). We conclude that SFRP5 gene methylation in leukemia cells activates Wnt/ß-catenin signaling to upregulate mdr1/P-gp expression and cause MDR. Recovery of SFRP5 expression reversed MDR in the KG1a leukemia cell line. Our results suggest that modulating SFRP5 methylation could decrease MDR in leukemia patients.
分泌型卷曲相关蛋白（SFRP）基因的甲基化激活Wnt/β-连环蛋白信号通路并促进肿瘤发展。本研究调查了SFRP5基因甲基化是否通过Wnt/β-连环蛋白信号通路导致白血病中的多药耐药（MDR），从而导致mdr1基因及其产物P-糖蛋白（P-gp）的上调。甲基化特异性PCR在12例急性白血病患者中7例的培养骨髓单个核细胞以及4种人白血病细胞系（HL-60、Raji、U937和KG1a）中鉴定出SFRP5基因甲基化。蛋白质印迹法显示，在5/7例SFRP5基因甲基化患者的细胞以及所有细胞系中均未检测到SFRP5蛋白表达。用去甲基化剂（DAC）处理可恢复SFRP5表达。在5例无SFRP5表达的患者中的3例细胞以及KG1a细胞系中检测到mdr1 mRNA和P-gp蛋白；这些细胞中活化的β-连环蛋白水平也最高。在这3例患者的细胞中，DAC恢复了SFRP5表达并下调了mdr1和P-gp。与KG1a/eGFP或未转染的KG1a细胞相比，转基因KG1a/SFRP5细胞中SFRP5蛋白表达得以恢复。KG1a/SFRP5细胞中的mdr1和P-gp被下调。KG1a/SFRP5细胞（0.573±0.131μM）的阿霉素IC50值显著低于KG1a细胞（0.963±0.115）或KG1a/eGFP细胞（0.917±0.138）（P<0.05）。我们得出结论，白血病细胞中的SFRP5基因甲基化激活Wnt/β-连环蛋白信号通路以上调mdr1/P-gp表达并导致MDR。SFRP5表达的恢复逆转了KG1a白血病细胞系中的MDR。我们的结果表明，调节SFRP5甲基化可降低白血病患者的MDR。
