The warburg effect in leukemia-stroma cocultures is mediated by mitochondrial uncoupling associated with uncoupling protein 2 activation

Abstract

In 1956, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect (i.e., the inhibition of anaerobic fermentation by oxygen). Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here, we show that under normoxic conditions exposure of leukemia cells to bone marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (DeltaPsiM) in both cell types. Notably, the consumption of glucose was not altered in cocultures, suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in DeltaPsiM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas small interfering RNA-mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment activation of highly conserved mammalian UCPs may facilitate the Warburg effect in the absence of permanent respiratory impairment.

Figure 1. Leukemia-MSC cocultures exhibit increased aerobic glycolysis

A) Leukemia cells (2.5–5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 48 h, and lactate levels and viable cell numbers were determined as described in the Methods. B) OCI-AML3 cells were cultured with MSC and absolute lactate levels were determined in the culture supernatants after 48 h; * = p<0.001 from MSC alone; **=p<0.005 from OCI-AML3 cells alone. C) Leukemia cells and MSC were cultured as in A) and glucose levels were monitored in the culture supernatants as described in the Methods. D) Cells cultured as above were analyzed by flow cytometry for uptake of the fluorescent glucose derivative 2-NBDG as described in the Methods. All experiments were done in replicates and repeated at least three times. * = p<0.05 from uncocultured controls for all experiments, except in B).

Figure 2. MSC decrease ΔΨM in leukemia cells

A) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 24 h followed by analysis of JC-1 fluorescene by flow cytometry as described in the Methods. B) HL60 and OCI-AML3 cells were cultured with MSC and analyzed as above; * = p<0.005 from uncocultured control. C) MSC cultured alone or with HL60 or OCI-AML3 cells were analyzed for ΔΨM as described in the Methods; * = p<0.001 from uncocultured controls.

Figure 3. MSC coculture does not reduce oxygen consumption or mitochondrial mass in leukemia cells

A) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) and oxygen consumption was determined after 24 h fluorometrically as described in the Methods; * = p<0.001 from uncocultured controls. B) The above cultures were also subjected to polarographic analysis. C) OCI-AML3 cells cultured alone or in the presence of MSC were FACS sorted based on CD90 expression and protein lysates were blotted with a cocktail of total OXPHOS complexes.

Figure 4. MSC induce mitochondrial uncoupling in leukemia cells

A) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 24 h followed by treatment with oligomycin (0–10 µM) for 60 minutes. Cells were then harvested and ΔΨM was quantitated as described in the Methods; * = p<0.05, ** = p<0.005 from uncocultured controls. B) Cells were cultured as above from 0 – 180 minutes and similarly analyzed for JC-1 fluorescence signals; * = p<0.05, ** = p<0.005 from uncocultured controls. C) OCI-AML3 and HL60 cells (5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 3 h followed by quantitation of viable cells and fluorometric oxygen consumption as described in the Methods; * = p<0.001 from uncocultured controls. D) OCI-AML3 and HL60 cells (5.0 × 10⁵ cells/ml) were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 48 h followed by FACS separation of leukemia cells and Western blotting as described in the Methods.

Figure 5. MSC induce the activation of UCP2

A) OCI-AML3 cells were cultured with feeder layers of MSC (5 × 10⁴ cells/ml) for 0, 30, 60, and 120 minutes followed by FACS separation of leukemia cells and Western blotting as described in the Methods B) OCI-AML3 cells were electroporated with siRNA duplexes targeting UCP2 or scrambled control (scr) duplexes as described in the Methods. 16 h after nucleofection cells were exposed to MSC feeder layers (5 × 10⁴ cells/ml) and ΔΨM and UCP2 protein levels were monitored 3 h after exposure as described in the Methods; * = p<0.05 from scr siRNA cocultured controls. Numbers below WB represent ratio of UCP2 to actin normalized to control. C) Cells were treated as above and lactate accumulation in the medium was quantitated after 48 h in coculture. D) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were treated with 2 µM CCCP for 48 h and lactate accumulation was quantitated as described in the Methods; * = p<0.001 from untreated controls.

Figure 6. Mitochondrial uncoupling in leukemia-MSC cocultues does not require de novo protein synthesis and may promote chemoresistance in leukemia cells

A) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were treated with 3.5 µM cycloheximide and exposed to MSC feeder layers for 3 h. ΔΨM was quantitated as described in the Methods. B) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were cultured with MSC feeder layers but separated by a 0.4 µm transwell filter for 24 h followed by quantitation of ΔΨM as above; ** = p < 0.001 from uncocultured control; * = p<0.05 from transwell cocultures C) OCI-AML3 cells (5.0 × 10⁵ cells/ml) were exposed to 1 and 2.5 µM CCCP for 30 minutes followed by the addition of MX (120 nM) for 24 h. % specific apoptosis was quantitated by flow cytometric analysis of Annexin V staining using the formula [(test – control)/(100 – control)]×100%; **= p<0.01 from MX treated OCI-AML3 cells. D) OCI-AML3 cells (2.5 × 10⁵ cells/ml) were cultured alone or in the presence of MSC feeder layers (5 × 10⁴ cells/ml) for 48 h in the presence of increasing concentrations of MX, AraC, or Vcr. Apoptosis was quantitated by flow cytometric analysis of Annexin V staining as above.

Figure 7. MSC cocultures do not prevent growth inhibitory effects of traditional chemotherapy

A) HL60 cells (2.5 × 10⁵ cells/ml) were cultured with MSC feeder layers (5 × 10⁴ cells/ml) and treated with increasing concentrations of MX for 48 h. Apoptosis was quantitated by flow cytometric analysis of Annexin V staining. B) OCI-AML3 cells were cultured as in D) and the number of viable cells was quantitated by trypan blue exclusion;* = p<0.05, ** = p<0.01 from cells alone.