Abstract
Acute myeloid leukemia (AML) is associated with numerous mutations, with the Feline McDonough Sarcoma (FMS) like tyrosine kinase 3 (FLT3) mutation resulting in with poor prognosis and outcome. Therapies have been developed using FLT3 inhibitors, however, drug resistance often leads to disease relapse. In this study, α-Mangostin and doxorubicin (Dox) were evaluated, singly and in combination, for their anti-leukemic effect on MOLM-13, an AML cell line with FLT3-ITD mutation. Cell viability and apoptosis were determined using CyQUANTGR and TUNEL assay, respectively. Cell cycle analysis was conducted on propidium iodide-stained cells using flow cytometry. Cellular proteins were quantified using Western blot technique, with additional study by ELISA for FLT3 kinase activity. The results revealed that cell treatment by the combined drug, Dox (1 µM) and α-Mangostin (20 µM), compared to Dox (1 µM) alone, caused a significant inhibitory effect (P<0.001) and indicated synergistic cell growth inhibition. The combined drug also showed increased TUNEL positive apoptotic cells and increased expression of the pro-apoptotic protein Bak compared to Dox alone (P<0.05). Dox treated cells, as well as the combined drug induced cell cycle arrest at G2/M phase compared to untreated cells (P<0.05 and P<0.001, respectively). There was also statistically significant (P<0.05) reduction of cdc25 phosphatases (enzymes which play an important role in G2/M transition) by the combination drug compared to sole cell treatment by Dox. Furthermore, phosphorylated FLT3 protein expression was reduced when the combined treatment was compared to Dox only after 2 h (P<0.05) and after 24 h (P<0.001). Thus, Dox and α-Mangostin combined treatment inhibited FLT3 phosphorylation in MOLM-13 cells which could have contributed to G2M cell arrest and apoptosis via cdc25s and Bak proteins respectively. Further studies are warranted to further evaluate the potential of Dox and α-Mangostin combined drug as inhibitors of FLT3-ITD phosphorylation and its potential clinical relevance in AML treatment.
Highlights
Acute Myeloid Leukemia (AML) is an aggressive blood cancer with fast progression and characterised by poor prognosis with treatment failure due to disease relapse
MOLM-13 derived from the peripheral blood of a relapsed acute myeloid leukemic patient with the classification FAB M5 and the FLT3 mutation, was originally a myelodysplastic syndrome which progressed to Acute myeloid leukemia (AML) [11]
To determine if cell death was via the induction of apoptosis, the morphological changes in MOLM-13 cells were compared between the vehicle control and drug-treated cells after 48 h treatment
Summary
Acute Myeloid Leukemia (AML) is an aggressive blood cancer with fast progression and characterised by poor prognosis with treatment failure due to disease relapse. After first line chemotherapy and stem cell transplant, most patients achieve remission, but about 40% develop disease relapse. The success of AML treatment is affected by recurrent genetic and cytogenetic alterations. Among these recurrent alterations, mutations of the Feline McDonough Sarcoma (FMS) such as tyrosine kinase 3 occurs most frequently [2]. FLT3 is a class III receptor tyrosine kinase that plays a crucial role in the proliferation of haematopoietic progenitor cells as well as their differentiation [2]. FLT3 activating mutation may occur either at the juxtamembrane domain (FLT3-ITD) or tyrosine kinase domain
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