Selective modulation of cell signalling by flavones in acute myeloid leukaemia and non-cancerous peripheral blood cells in vitro - preliminary study

Background:
Established chemotherapy drugs for Acute Myeloid Leukaemia (AML) such as doxorubicin (Dox) are known to cause adverse side effects. Additionally, AML-associated mutations and microenvironment contribute to the development of drug resistance and poor prognosis, presenting further challenges to treatment. Flavones such as baicalein (BL) possess anticancer properties, with the potential as adjuvant drugs with favourable safety.
Aim:
The aim of this preliminary study was to determine half maximal cell inhibitory concentrations of selected flavones and Dox and investigate possible mechanisms of action.
Methods:
CyQuantGR cell cytotoxicity assay was conducted using AML cell models: MOLM-13 (relapsed leukaemia) and U937 (non-relapsed), in addition to a non-cancerous B-lymphocyte cell line, RPMI1788. Annexin V/propidium iodide double staining was used to assess the induction of cell death using Flow Cytometry. Flow cytometry was also used to assess 72 h-cell proliferation rate with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) stained cells.
Results
For all cell lines, a dose-dependent relationship was observed between cell viability and Dox treatments (0.039 μM - 2.5 μM) with AML-MOLM-13 showing 1.5-fold lower IC50 (=0.034 μM; p=0.086) and AML-U937 showing 2-fold higher IC50 (=0.131 μM, p<0.001) than non-cancer-RPMI1788 (IC50=0.050 μM). For the
flavones (Flavone 1 (FL1), 2 (FL2) and BL) tested at (1.25 μM – 80 μM) in MOLM-13, FL1 and FL2 demonstrated dose-dependent cytotoxicity. However, cytotoxicity was observed for BL only at concentrations > 10 μM. In U937, tested flavones significantly (p<0.05) decreased cell viability at concentrations >20 μM (BL) and >40 μM (FL1-2). In RPMI1788, dose-dependent cytotoxicity was observed if [flavone] >20 μM. BL was 3-fold more cytotoxic (p<0.001) in MOLM-13 (IC50=19 μM) and 1.5-fold more cytotoxic (p<0.001) in U937 (IC50=39 μM) than RPMI1788 (IC50=59 μM). Similarly, for FL1, MOLM-13 was 8-fold (IC50=9 μM; p<0.001) and U937 was 1.2-fold (IC50=56 μM; p<0.001) more sensitive to the flavone than RPMI1788 (IC50=72 μM). FL2 demonstrated IC50=16 μM in MOLM-13, but in RPMI1788 and U937 the FL2 did not reduce cell viability below 50%. In the non-relapsed AML cells (U937), cell death studies demonstrated that BL 40 μM significantly reduced live cell population (p=0.041) and increased the population of necrotic cells (p=0.022) after 48h model but no significant findings were observed for the other compounds. Similarly, BL 40 μM
significantly increased the doubling time of U937 (p<0.05) and MOLM-13 (p<0.05) CFSE stained cells compared to vehicle control.
Conclusions:
Data from this study suggests the flavones may selectively inhibit relapsed AML more effectively than the non-cancerous cells in vitro. BL (40 μM) is the only compound that significantly increased necrotic cells population in U937 AML cells, a possible mechanism of cell death induction apart from apoptosis. BL also demonstrated decrease in cell proliferation from the CFSE data, suggesting another possible mechanism of
action. Further studies are underway to assess the structure-related activities of the flavones and drug combinations. Using in vitro and in silico methods, future work will aim to evaluate the cell signalling pathways such as apoptosis, autophagy, and Wnt to further assess the mechanisms of action at protein, genetic and epigenetic levels.