Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Japan.
Exp Hematol. 2010 Aug;38(8):666-76. doi: 10.1016/j.exphem.2010.03.020. Epub 2010 Apr 9.
Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D(3) inhibited the proliferation of MCF-7 breast cancer cells in association with inhibition of the mammalian target of rapamycin (mTOR) signaling. This study explored the drug interaction of 1,25(OH)(2)D(3) and rapamycin analog RAD001 (everolimus) in AML cells.
Effects of RAD001 and 1,25-(OH)(2)D(3) on the proliferation and differentiation of U937 cells were assessed by colony-forming assay and quantification of CD11b cell surface antigens and their endocytic capability, respectively. Effects of RAD001 and 1,25-(OH)(2)D(3) on Akt/mTOR complex-1 (mTORC1) signaling and cell-cycle-related molecules were explored by Western blot analysis. The reporter gene and chromatin immunoprecipitation assays were employed to examine the effects of RAD001 and 1,25-(OH)(2)D(3) on the promoter of the p21(waf1) gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)(2)D(3) in vivo.
RAD001 potentiated the ability of 1,25(OH)(2)D(3) to induce growth arrest and differentiation of AML cells in parallel with downregulation of the levels of p-S6K and p-4E-BP1, substrates of mTORC1. In addition, RAD001 significantly enhanced 1,25(OH)(2)D(3)-mediated transcriptional activity of p21(waf1) in association with increased levels of the acetylated forms of histone H3 and vitamin D receptor bound to the p21(waf1) promoter in U937 cells. Moreover, RAD001 (3 mg/kg, every another day) significantly enhanced 1,25(OH)(2)D(3)-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects.
Concomitant administration of 1,25(OH)(2)D(3) and the mTORC1 inhibitor may be a promising treatment strategy for individuals with AML.
通过使用 1,25-二羟维生素 D（1,25(OH)(2)D(3)）等诱导分化剂进行分化诱导治疗是治疗急性髓性白血病（AML）的一种有效方法。我们之前的研究表明，Gemini-23-yne-26,27-六氟-D（3）可抑制 MCF-7 乳腺癌细胞的增殖，同时抑制哺乳动物雷帕霉素靶蛋白（mTOR）信号。本研究探讨了 AML 细胞中 1,25(OH)(2)D(3)和 rapamycin 类似物 RAD001（依维莫司）的药物相互作用。
通过集落形成试验和定量检测 CD11b 细胞表面抗原及其内吞能力，评估 RAD001 和 1,25-(OH)(2)D(3)对 U937 细胞增殖和分化的影响。通过 Western blot 分析研究 RAD001 和 1,25-(OH)(2)D(3)对 Akt/mTOR 复合物-1（mTORC1）信号和细胞周期相关分子的影响。采用报告基因和染色质免疫沉淀试验检测 RAD001 和 1,25-(OH)(2)D(3)对 p21(waf1)基因启动子的影响。利用 U937 鼠异种移植模型研究 RAD001 和 1,25-(OH)(2)D(3)在体内的作用。
RAD001 与下调 mTORC1 的底物 p-S6K 和 p-4E-BP1 的水平平行增强了 1,25(OH)(2)D(3)诱导 AML 细胞生长停滞和分化的能力。此外，RAD001 显著增强了 1,25(OH)(2)D(3)介导的 U937 细胞中 p21(waf1)的转录活性，与组蛋白 H3 的乙酰化形式和与 p21(waf1)启动子结合的维生素 D 受体水平增加有关。此外，RAD001（3mg/kg，每隔一天）在裸鼠中显著增强了 1,25(OH)(2)D(3)诱导的 U937 肿瘤异种移植物的生长抑制作用，且无不良反应。
联合使用 1,25(OH)(2)D(3)和 mTORC1 抑制剂可能是治疗 AML 患者的一种有前途的治疗策略。
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