In this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with doxorubin (Dox) and betulinic acid (BetA), singly and in combination. CyQUANT Direct^® and Annexin V/propidium iodide double staining were used to measure the cytotoxic and cell death induction effects of the compounds, respectively. Reactive oxygen species (ROS) generation was measured using 2′,7′-dichlorofluorescin diacetate staining. Expressions of proteins and genes were examined by Western blot and reverse transcription polymerase chain reaction analysis, respectively. BetA (20 μM) and Dox (1 μM) indicated a synergistic growth inhibitory effect on MOLM-13 cells. The combined drug caused more cells to reside in irreversible late apoptotic stage compared to the single treatments (p < 0.05). Elevation in ROS may be the synergistic mechanism involved in MOLM-13 cell death since ROS can directly disrupt mitochondrial activity. In contrast, in leukaemic U-937 cells, the combination treatments attenuated Dox-induced cell death. Dox and the drug combination selectively reduced (p < 0.05) a recently reported anti-apoptotic Bcl-2 protein isoform p15-20-Bcl-2 in MOLM-13 by our group, without affecting the usually reported p26-Bcl-2-α. Further studies using known inhibitors of apoptosis are required to confirm the potential of Dox-BetA combination to modulate these pathways.

中文翻译：

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桦木酸-阿霉素-药物组合在复发性 AML MOLM-13 细胞模型中通过 ROS 刺激诱导细胞凋亡

在这项研究中，用阿霉素 (Dox) 和桦木酸 (BetA) 单独和组合研究了来自复发性 MLL 重排细胞模型 (MOLM-13) 的 AML 细胞系中的细胞死亡调节和诱导。的CyQuant直接^®和Annexin V/碘化丙啶双重染色分别用于测量化合物的细胞毒性和细胞死亡诱导作用。使用 2',7'-二氯荧光素二乙酸酯染色测量活性氧 (ROS) 的产生。分别通过蛋白质印迹和逆转录聚合酶链反应分析检测蛋白质和基因的表达。BetA (20 μM) 和 Dox (1 μM) 表明对 MOLM-13 细胞具有协同生长抑制作用。与单一治疗相比，联合药物导致更多细胞处于不可逆的晚期凋亡阶段（p< 0.05)。ROS 的升高可能是参与 MOLM-13 细胞死亡的协同机制，因为 ROS 可以直接破坏线粒体活性。相比之下，在白血病 U-937 细胞中，联合治疗减弱了 Dox 诱导的细胞死亡。Dox 和药物组合选择性减少（p < 0.05）我们小组最近报道的 MOLM-13 中的抗凋亡 Bcl-2 蛋白同种型 p15-20-Bcl-2，而不影响通常报道的 p26-Bcl-2-α . 需要使用已知的细胞凋亡抑制剂进行进一步研究，以确认 Dox-BetA 组合调节这些途径的潜力。