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Adaptation of Leukemia Cells to Hypoxic Condition Through Switching the Energy Metabolism or Avoiding the Oxidative Stress

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Adaptation of Leukemia Cells to Hypoxic Condition Through Switching the Energy Metabolism or Avoiding the Oxidative Stress

Mineaki Goto et al. BMC Cancer.

Abstract

Background: Like normal hematopoietic stem cells, leukemia cells proliferate in bone marrow, where oxygen supply is limited. However, the growth and energy metabolism of leukemia cells under hypoxia have not been well understood. Although it has been known that reactive oxygen species (ROS) is generated under hypoxic conditions, normal and leukemia stem cells were characterized by relatively low levels of ROS. Roles of ROS on leukemia cells under hypoxia also have not been well understood.

Methods: Four Leukemia cell lines were cultured under normoxia (21% O2) or hypoxia (1% O2), where NB4 and THP-1 were most extensively studied. To evaluate energy metabolism, we estimated whole cell number or apoptotic cells with or without a glycolysis inhibitor or an oxidative phosphorylation (OXPHOS) inhibitor. Glucose consumption and lactate production were also measured. To evaluate oxidative stress in hypoxic condition, the ROS level and GSH (reduced glutathione) / GSSG (oxidized glutathione) ratio was measured. In addition, pyruvate dehydrogenase kinase 1 (PDK1) and cytochrome c oxidase subunit 4 (COX4) were examined by western blotting or RT-PCR.

Results: NB4, which grows well under normoxia depending on glycolysis, demonstrated prominent apoptosis and growth suppression after 48 hours culture under hypoxia. NB4 cells cultured under hypoxia showed significantly increased ROS. Culture with a ROS scavenger resulted in decrease of apoptotic cell death of NB4 under hypoxia. NB4 cells cultured for longer period (7 days) under hypoxia did not come to extinction, but grew slowly by upregulating GSH synthesis to protect from ROS generated in hypoxic condition. By contrast, THP-1, which largely depends on OXPHOS in mitochondria under normoxia, demonstrated more growth under hypoxia by changing metabolism from OXPHOS to glycolysis through upregulating PDK1. Moreover, THP-1 avoided ROS generation by substituting COX 4 subunit (from COX 4-1 to COX 4-2) through upregulation of LON, a mitochondrial protease under hypoxia.

Conclusions: We showed that leukemia cells survive and adapt to the hypoxic condition through various pathways. Our results will help understanding energy metabolism of leukemia cells and creating novel therapeutics.

Figures

Figure 1
Figure 1
Cell proliferation of four leukemia cell lines with no treatment under normoxia or hypoxia. 2 × 105/ml cells in 0.5 ml RPMI1640 + 3% FCS were cultured under normoxia (21% O2) or hypoxia (1% O2) for 48 hours in triplicate. Then, cell count was measured after 24 and 48 hours, respectively. NB4 (A) significantly diminished their proliferation under hypoxia compared with normoxia (48 hours) (p = 0.0001). In contrast, THP-1 (B) significantly increased more under hypoxia than normoxia (48 hours) (p = 0.005). Kasumi-1 (C) and HL-60 (D) did not show significant difference of cell growth between normoxia and hypoxia (48 hours). ▲: under hypoxia, ●: under normoxia. *** p < 0.001, ** p < 0.01, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 2
Figure 2
Apoptosis assay by annexin V staining under hypoxia versus normoxia on NB4 and THP-1. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 24 hours in triplicate. Then, apoptotic cells were examined after 12 and 24 hours by annexin V / PI method. Apoptotic cells of NB4 (A) under 24 hours hypoxia were significantly increased (p = 0.0031), although THP-1 (B) did not show increased apoptosis under hypoxia at least 24 hours (p = 0.7876). ▲: under hypoxia, ●: under normoxia. H: under hypoxia, N: under normoxia. *** p < 0.001, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 3
Figure 3
ROS and its effects on the viability of NB4 and THP-1 under hypoxia. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 48 hours with or without NAC 0.5 mM. ROS levels were measured by using CellROX Deep Red Reagent. The viable cell numbers of NB4 and THP-1 with or without NAC 0.5 mM were examined by annexin V / PI method in triplicate. ROS level of NB4 (A) was much higher under 48 hours hypoxia (red line) than normoxia (black line). Incubation with NAC made ROS level decreased in NB4 under hypoxia (blue line). NAC treatment increased viable cells cultured under hypoxia (p = 0.0002) (B right), although the effect of NAC was not so obvious under normoxia (p = 0.0285) (B left). On the other hand, THP-1 did not show ROS increase under hypoxia and addition of NAC did not work as same as NB4 (C, D). black line: ROS level under normoxia as control, red line: under hypoxia, blue line: under hypoxia with NAC. H: under hypoxia, N: under normoxia. *** p < 0.001, * p < 0.05, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 4
Figure 4
GSH / GSSG ratio measurement under normoxia and two different terms of hypoxia. 2 × 105/ml cells in 0.02 ml RPMI1640 + 3% FCS were cultured under normoxia (21% O2) or hypoxia (1% O2) (short term hypoxia) for 24 hours in triplicate. Then, GSH / GSSG ratio was measured by using GSH / GSSG-Glo Assay. After 7 days culture under hypoxia with two medium changes, GSH / GSSG ratio was also measured by using these cells cultured for 24 more hours under hypoxia (long term hypoxia). GSH / GSSG ratio of NB4 under normoxia was 3.02 ± 0.067 and that under short term hypoxia was 2.31 ± 0.020 (p = 0.0005). GSH / GSSG ratio under long term hypoxia (2.69 ± 0.028) was significantly higher than that of short term hypoxia (p = 0.0004). GSH / GSSG ratio of THP-1 under normoxia (1.50 ± 0.094) and hypoxia (1.50 ± 0.083) was almost equal. THP-1 under longer hypoxia showed increase of GSH / GSSG ratio (2.09 ± 0.077) compared with that under short term hypoxia (p = 0.0065). SH: under short term hypoxia, LH under long term hypoxia, N: under normoxia. *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 5
Figure 5
Glucose consumption and lactate production in NB4 and THP-1 under normoxia versus hypoxia. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 48 hours in triplicate. Then, concentration of glucose and lactic acid in the culture supernatant was measured by using D-Glucose kit or L-Lactic acid kit. Both cell lines showed increased glucose consumption under hypoxia compared with normoxia with statistical significance (p = 0.0013 in NB4, p = 0.0054 in THP-1) (A). THP-1 demonstrated increased lactate production under hypoxia (p < 0.0001), but NB4 did not (p = 0.0663) (B). H: under hypoxia, N: under normoxia. *** p < 0.001, ** p < 0.01, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 6
Figure 6
Growth inhibition by 2-FDG or oligo on NB4 and THP-1 under normoxia and hypoxia. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 48 hours with 2-FDG (0, 2, 5 10 mM) or oligo (0, 0.05, 0.2, 1 μg/ml) in triplicate. The cell numbers after 48 hours culture under normoxia or hypoxia with 2-FDG or oligo were divided by cell numbers of control (average of triplicated experiments) to obtain cell number ratios, in which the cell number with no treatment corresponded to 100%. The growth suppression of THP-1 by 2-FDG even at 2 mM under hypoxia was significantly more pronounced than that under normoxia (p = 0.0005), although the growth inhibition of NB4 by 2-FDG under hypoxia was slightly less than that under normoxia (A). Oligo did not suppress the growth of NB4 under hypoxia, but suppressed that under normoxia (at 1 μg/ml, p < 0.0001). Oligo similarly suppressed the growth of THP-1 under normoxia and hypoxia (B). ▲: under hypoxia, ●: under normoxia. *** p < 0.001, ** p < 0.01, ns p > 0.05 (unpaired, two-tailed t-test).
Figure 7
Figure 7
Western blotting of PDK1. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 24 hours. PDK1 was abundantly expressed in NB4 even under normoxia, culture under hypoxia did not augment the expression of PDK1 in NB4. Expression of PDK1 was augmented by hypoxia in THP-1. Top, PDK1; bottom, β-actin. H: under hypoxia, N: under normoxia.
Figure 8
Figure 8
RT-PCR analysis of COX 4–1 and LON on NB4 and THP-1 under normoxia or hypoxia. NB4 and THP-1 were cultured in the same condition as cell proliferation assay for 24 hours. The expression of COX 4–1 in THP-1 under hypoxia was decreased compared with normoxia. In addition, mRNA expression of LON in THP-1 under hypoxia was increased compared with that in normoxia. In NB4, mRNA expression of COX 4–1 and LON was not influenced by O2 concentration. β-actin was performed as an internal control. Top, COX 4–1; middle, LON; bottom, β-actin. H: under hypoxia, N: under normoxia.

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