Doxorubicin combined with phytochemicals as pro-apoptotic agents and protein kinase inhibitors for the treatment of acute myeloid leukaemia

Acute myeloid leukaemia is one of the most common form of adult leukaemia with several kinase mutations. The FLT3-ITD mutation is a common kinase mutation observed in (20-30%) of AML patients and this mutation is also the most involved in the prognosis of AML. It confers poor treatment outcome in patients despite intensive chemotherapy due to an increased relapse rate. Kinases are crucial in controlling different cellular activities, and their mutations are well known to signal cancer development and progression. FLT3 inhibitors have been evaluated in clinical trials leading to the approval of Midostaurin for the treatment of AML. However, drug resistance and kinase selectivity are still challenges faced by current FLT3 inhibitors. The aim of this study was to investigate in vitro, the anti-leukaemic effect of phytochemicals that are commonly consumed and widely distributed in plants and fruits (α-mangostin, gallic acid and vitamin C), singly and in combination with doxorubicin (Dox) in the relapsed AML cell line MOLM-13. CyQUANT proliferation assay was used to determine their effects on cell viability. Flow cytometric analysis of cell apoptosis and cell cycle were conducted. Expression of various proteins (Bax, Bak, Bcl-2, caspase 3, 8 and 9, p53, p21, p16, cdc25s and FLT3) were determined using Western blot technique, and ELISA to confirm FLT3-ITD phosphorylation. Statistical analysis including ANOVA, Student T-test were chosen for comparing the effects of the standard chemotherapeutic drug (Dox) with the phytochemicals, either singly used or combined. Results showed that α-mangostin at the concentration 5-50 μM inhibited cell growth in a dose-dependent manner in MOLM-13 cell line. Gallic acid and vitamin C inhibited cell growth at 5-30 μM. More apoptotic effects were observed when α-mangostin (20 μM) was combined with Dox (1 μM), with more TUNEL positive cells and increased expression of the pro-apoptotic protein Bak. Treatments of vitamin C (15 μM) combined with Dox (1 μM) induced early apoptotic cell death when compared to Dox only. Gallic acid (15 μM) showed synergistic effect with Dox (1μM) in suppressing cell growth and induced apoptosis was associated with the expression of Bak (p<0.01). However, Dox only and vitamin C single treatment significantly inhibited Bak (p<0.01) in this study. Furthermore, combination of either α-mangostin, gallic acid or vitamin C and Dox blocked MOLM-13 cells at G2/M phase, indicating irreversible cell cycle arrest. Induction of senescence, an anti-tumorigenic mechanism that inhibit cancer was determined by p16 expression to suggest senescence. Reactivation of senescence pathway results in cells that should not undergo repair in the cell cycle and will not divide even in the presence of mitotic factors. In this study, expression of p16 protein by combination of doxorubicin with the phytochemicals was not significant when compared to Dox only, while vitamin C and Dox combination was significant compared to negative control (p<0.05). In addition, doxorubicin combined with α-mangostin suppressed phosphorylation of FLT3-ITD and cdc25s phosphatases significantly (p<0.05). In conclusion, results from this study suggests that the mechanism of drug action of Dox, when combined with phytochemicals is through the induction of apoptosis resulting in irreversible programmed cell death Further studies revealed that α-mangostin had a synergistic effect with doxorubicin in the inhibition of phosphorylated FLT3-ITD which could have promising potential to be developed as FLT3 inhibitors. Therefore, AML cells treated with a combination of α-mangostin and Dox promotes the induction of apoptosis that could be effective to overcome resistance in AML and therefore require further study to understand its mechanism as a targeted FLT3-ITD therapy.