Induction of death in myeloid leukaemia cells by doxorubicin and betulinic acid, singly and in combination was associated with the regulation of apoptosis, autophagy and the PI3K/Akt pathways

PhD thesis


Vu, M. 2020. Induction of death in myeloid leukaemia cells by doxorubicin and betulinic acid, singly and in combination was associated with the regulation of apoptosis, autophagy and the PI3K/Akt pathways. PhD thesis Middlesex University Science and Technology
TypePhD thesis
TitleInduction of death in myeloid leukaemia cells by doxorubicin and betulinic acid, singly and in combination was associated with the regulation of apoptosis, autophagy and the PI3K/Akt pathways
AuthorsVu, M.
Abstract

Relapsed acute myeloid leukaemia (AML) cells are difficult to eradicate due to their more refractory nature. Deregulation of the B-cell lymphoma 2 (Bcl-2) family of proteins in these cells, which regulates cell death through apoptosis, autophagy and other cell processes, may contribute to this trend. Using chemical agents could modulate these signalling pathways. Drugs such as doxorubicin (Dox) are effective but displayed some clinical drawbacks, while compounds such as betulinic acid (BetA) have shown some interesting activity against cancerous cells. These drugs may complement each other in sensitising and eradicating aggressive AML cells. Therefore, in this study, cell death regulation and induction in AML cell line from a relapsed MLL-rearranged cell model (MOLM-13) was investigated with an established chemotherapy drug Dox and a bioactive compound BetA, singly and in combination.
The cytotoxic effect of the compounds was measured by CyQUANT Direct® (which estimates cell numbers by nuclear content of viable cells). Proliferation rate (using 5(6)- carboxyfluorescein diacetate succinimidyl ester) and cell death population (using Annexin V/propidium iodide) of the treatments were studied using flow cytometry. The contribution of reactive oxygen species (ROS) stimulation in MOLM-13 cell death mechanism was measured by 2’,7’–dichlorofluorescin diacetate (DCFDA) staining. Protein and gene expressions of regulators involved in apoptotic, autophagy and cell-survival (PI3K-AKT) pathways were examined by Western blot and reverse transcription polymerase chain reaction (RT- PCR) analysis, respectively.
Dox showed non-discriminatory inhibition of cell viability (p < 0.05) in all tested cell lines including non-cancerous (HEK293T) cell control whilst BetA selectively inhibited viability (p < 0.05) of AML MOLM-13 and CML K562. The combination index (CI; which determines multiple drug effect interactions) of BetA (20 μM) and Dox (1 μM) showed that the inhibitory effect on AML cells was synergistic (CI < 1). Dox potently inhibited cell division of MOLM-13 cells over a three-day period, which was not negatively affected by co-treatment with BetA combination. Although Dox inhibited (p <0.05) PI3K-AKT markers in MOLM-13 cells, suggesting a decrease in cell survival, potential sign of drug resistance was observed possibly due to significant downregulation (p <0.05) of PTEN. BetA-Dox combined treatments increased (p < 0.01) ROS formation within MOLM-13 cells between 0.5-1.5 h but the single treatments did not significantly alter ROS levels. Additionally, more cells resided in irreversible late apoptotic stage after drug combination (BetA + Dox) in MOLM-13 co-treatment compared to the single treatments (p < 0.05). However, a contradicting effect was observed in leukaemic SC/U-937 monocytes, where combination treatments attenuated Dox-induced cell death. Here for the first time an isoform of anti-apoptotic Bcl-2 protein p15-20-Bcl-2 was detected in MOLM-13, alongside the usual p26-Bcl-2-α. This p15-20-Bcl-2 was sensitive to treatments and was reduced (p < 0.05) upon exposure to Dox and the drug combination, unlike p26-Bcl-2-α present in all tested cells. Dox and the drug combination reduction in Beclin 1 (autophagy inducer) expression could indicate a modulation of autophagy in MOLM-13 cells but it is not clear whether this confers a protective or cell-death inducing effect. The outcome of combination studies on Bcl-2 family and autophagy protein expressions was in parallel with the mRNA regulation; additionally, the transcript further postulated the protective nature of BetA combination in Dox-treated SC/U-937 cells. Further recommended studies should investigate the targeting potential and the role of the novel p15-20-Bcl-2 and define the underlying cell-dependent effect mechanism of the combination treatments in enhancing and alleviating cell death.

Sustainable Development Goals3 Good health and well-being
Middlesex University ThemeHealth & Wellbeing
LanguageEnglish
Department nameScience and Technology
Institution nameMiddlesex University
Publication dates
Print09 Aug 2022
Publication process dates
Deposited09 Aug 2022
Accepted31 Jul 2020
Output statusPublished
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