Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, China.
J Exp Clin Cancer Res. 2010 Nov 18;29(1):150. doi: 10.1186/1756-9966-29-150.
Glucocorticoid (GC) resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL). In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex) treatment.
Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay. Fluorescence-activated cell sorting (FACS) analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR), the cell cycle regulatory proteins, and apoptosis associated proteins.
10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK) inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR) expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1.
Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting new therapeutic approach for GC resistant T-ALL patients.
糖皮质激素（GC）耐药经常发生在 T 细胞谱系急性淋巴细胞白血病（T-ALL）中。在这项研究中，我们研究了使用雷帕霉素恢复 GC 耐药 T-ALL 细胞对地塞米松（Dex）治疗敏感性的潜力和机制。
通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法检测细胞增殖。使用荧光激活细胞分选（FACS）分析来分析细胞凋亡和细胞周期。通过 Western blot 分析来检测哺乳动物雷帕霉素靶蛋白（mTOR）的下游效应蛋白、细胞周期调节蛋白和凋亡相关蛋白的表达。
10 nM 雷帕霉素显著增加了 GC 耐药 T-ALL 细胞对 GC 的敏感性，这种作用至少部分是通过抑制 mTOR 信号通路介导的。细胞周期停滞与 G1-S 期调节蛋白的调节有关。雷帕霉素和 Dex 都可以诱导细胞周期蛋白依赖性激酶（CDK）抑制剂 p21 和 p27 的上调，并且雷帕霉素与 Dex 的联合治疗导致它们的表达协同诱导。雷帕霉素对细胞周期蛋白 A 的表达没有明显影响，而 Dex 诱导细胞周期蛋白 A 的表达。雷帕霉素阻止 Dex 诱导的细胞周期蛋白 A 表达。雷帕霉素对细胞周期蛋白 D1 的抑制作用强于 Dex。雷帕霉素增强了 GC 诱导的细胞凋亡，而这不是通过调节糖皮质激素受体（GR）的表达来实现的，而是通过协同上调促凋亡蛋白如 caspase-3、Bax 和 Bim，以及下调抗凋亡蛋白 Mcl-1 来实现的。
我们的数据表明，雷帕霉素可以有效地逆转 T-ALL 中的 GC 耐药，这种作用是通过诱导细胞周期停滞在 G0/G1 期和激活内在凋亡程序来实现的。因此，mTOR 抑制剂雷帕霉素与含 GC 的方案联合使用可能是 GC 耐药 T-ALL 患者有吸引力的新治疗方法。
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