Betulinic Acid-Doxorubicin-Drug Combination Induced Apoptotic Death Via Ros Stimulation In A Relapsed Aml Molm-13 Cell Model

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(R) 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 mu M) and Dox (1 mu M) 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-alpha. Further studies using known inhibitors of apoptosis are required to confirm the potential of Dox-BetA combination to modulate these pathways.