Abstract
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.
Highlights
In our recent publication we reported that Dox exhibited selectivity toward relapsed acute myeloid leukaemia (AML) cells, associated with reduction of p15-20-B-cell lymphoma 2 (Bcl-2) and Beclin 1 protein expression in MOLM-13 cells [22]
We report that Dox induced cell death in MOLM-13 cells, with a decrease in the expression of Beclin 1 protein (Figure 4d)
Increases in Reactive oxygen species (ROS) generation by the combination treatment could be a mechanical factor that enhanced cell death, resulting in drug synergism in MOLM-13 cells, which led to an increase in more cells undergoing late apoptosis
Summary
Betulinic acid (3β,hydroxyl-lup-20(29)-en-28-oic acid, BetA), pentacyclic triterpenoid, has been reported to be effective against relapsed acute myeloid leukaemia (AML) cells when tested alongside standard AML chemotherapeutics such as doxorubicin (Dox) and cytarabine (tested at clinically relevant concentrations) [1]. It has been reported that BetA exerts some selectivity towards cancer cells whilst being less toxic towards non-cancerous cells. This effect has been described in both blood cells and cells of epithelial origin, such as human primary melanocytic cells [2], normal colon cells [3], peripheral blood mononuclear cells [4], and normal peripheral blood lymphoblasts [5]
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