Acute myeloid leukemia has a high rate of relapse, partly because of the existence of chemo- and radio-therapy-resistant leukemia stem cells. Sensitization of LSCs is an active research area [20]. The metabolic plasticity of cancer stem cells is thought to contribute to their resistance indicating the importance of mitochondria [21][22]. Aiming to sensitize leukemia stem-like cells i.e., CD34+, the effects of Ara-c, 2-DG, and their combination were assessed by MTT, ROS, and MMP.
Results showed that Ara-c increases the ROS in order of CD34+ < KG1-a < CD34− indicating the ability of the stem-like cell, CD34+, to avert ROS induction by Ara-c. LSCs depend on OXPHOS as an energy source which is ROS productive. LSCs use various mechanisms such as mitophagy, glutathione, or antioxidants to keep ROS low which is an important hallmark of stem cells [23]. Ara-c induces ROS production through inhibition of DNA polymerase gamma, and hence induces cellular stress by disruption of mitochondrial function and ROS increment [24][25]. Cells that have more myeloperoxidase, an antioxidant enzyme, are more resistant to Ara-c than other cell lines [26]. While most of the reports indicate the lower ROS content in chemo-resistant cells, Jean-Emmanuel Sarry et al., could not find any correlation between Ara-c resistance and ROS content [27]. In personal contact with author Jean-Emmanuel Sarry, he assigned this controversy to a low Ara-c dose and an in-vivo study conducted by his group.
2-DG could boost ROS in all groups, but this enhancement was not much in CD34+ cells. 2-DG can enhance ROS by reducing glucose metabolism [28]. One of the reasons for CD34+ resistance to 2-DG could be the high contents of hexokinase II in stem-like cells [29]. Similar results were reported on liver cancer stem cell metabolism. Liu et al. showed that 2-DG elevates ROS and OXPHOS in high glycolysis and OXPHOS liver cancer stem cells [30]. However, there are some reports in which 2-DG did not change [31], sometimes even decreased, ROS production in AML-derived cell lines [32]. According to our knowledge and personal communication with some authors, we believe that such contradictions may arise from variations in studied cells or culture medium. For instance, KG1-a is more dependent on glycolysis compared to Hl-60 [8] and hence KG1-a would be more susceptible to increased ROS upon 2-DG treatment. Culture mediums may differ in the amount of pyruvate i.e., culture medium with high pyruvate could not affected by 2-DG [33].
Mitochondria membrane potential (MMP) is important for mitochondrial ATP production and is controlled by gene expression [34]. Increased ROS by Ara-c treatment leads to mitochondrial outer membrane permeability, followed by MMP reduction, and finally apoptosis [35]. Similarly, we found that Ara-c reduced MMP, albeit the MMP change in CD34+ cells was not significant. Along with our results, AML CD39+ resistance cells also showed resistance to MMP loss by Ara-c through the cAMP-mediated mitochondrial adaptive stress response [27]. It was also shown that cells containing low myeloperoxidase, e.g., MOLM-14, are more affordable to MMP loss by Ara-c [36]. In contrast to our study, investigations on AML cells obtained from patients illustrated related MMP resistance to oxidative phosphorylation instead of stemness of leukemia cells [35]. Mercier et al, showed that Ara-c did not affect the MMP of AML cells, which could be due to cell source, cell type, and Ara-C dosage [37].
2-Deoxy-D-glucose (2-DG) can reduce mitochondrial membrane potential by inhibiting glucose metabolism [38]. We observed that 2-DG caused MMP loss in CD34+, CD34−, and crude KG1-a cells though CD34+ cells were less affected. The less affectedness of CD34+ cells by 2-DG may arise from their metabolic plasticity i.e., shifting to OXPHOS [39]. Studies on co-treatment of metformin and 2-DG on breast cancer stem cells indicated that 2-DG decreased MMP in these cells. 2-DG increases mitochondrial depolarization in mutated mtDNA cells by selective inhibition of mtDNA compared to control cells [38].
As mentioned, both Ara-c and 2-DG enhanced ROS in CD34+, CD34−, and KG1-a cells, but CD34+ cells were more resistant to ROS alteration. The combination of these drugs was used to sensitize CD34+ cells. Results illustrated that the combination of a high dose of Ara-C with ½ EC50 dose of 2-DG resulted in a significant boost in ROS compared to each drug alone. Many reports indicated that a combination of drugs could increase the effectiveness for example; the combinations of myeloperoxidase inhibitor and Cytarabine in leukemia cells [26], EIF4A inhibitor and Ara-c in MOLM-14 cell line [37], 2-DG with arsenic trioxide in AML derived cell line [40] and 2-DG and with l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in glioblastoma cells [41]. However, there are some controversies to this for example combining Exotimer with 2-DG on HL-60 cells did not increase ROS [31]. Different responses to combination therapy in various cell lines could attributed to their dependency on different metabolic pathways [42].
Also, our studies indicated that the Ara-c/2-DG combination reduced MMP in all cell groups more than in each alone. Previous studies have observed that Ara-c accelerates the loss of mitochondrial membrane potential when combined with another drug or inhibitor, for example in co-treatment of MOLM-14 cells with myeloperoxidase inhibitor (ABAH) and Ara-c causes greater loss of MMP [26]. Furthermore, in another study on a chemotherapy-resistant AML cell, CD39+, the combination of CD39 expression inhibitor (H89) and Ara-c resulted in a further loss of mitochondrial membrane potential [11]. 2-DG in combination with metformin decreased mitochondrial membrane potential [43].
Metabolic reprogramming is a promising strategy to combat chemotherapy-resistant cells. The metabolic structure of leukemia stem cells is flexible and allows them to survive in stressful conditions and evade chemotherapy harm. Metabolic changes are associated with mitochondrial dysfunction, which affects various cellular functions such as proliferation, apoptosis, anabolism, and autophagy [21][22]. The current study explored the effect of Ara-c, 2-DG, and their combination on ROS and MMP in stem-like cells and showed that the combination could prevent metabolic flexibility in LSCs.