Introduction

Reprogramming energy metabolism is an emerging target of cancer. Glycolysis is frequently upregulated in highly proliferative cancer cells because it provides biosynthetic advantage for growth. Dichloroacetate (DCA), a PDK inhibitor currently tested in the clinics, is known to shift metabolism from glycolysis to glucose oxidation and to decrease the proliferation in various cancer cells lines¹. However, as tumor cells are presenting extraordinary metabolic plasticity, PDK inhibition may lack efficacy because of the induction of compensatory mechanisms sustaining tumor growth. Moreover, this exacerbated glycolytic flux in cancer cells leads to extracellular tumor acidosis². Conversely, little is known about how tumor cells adapt their metabolism to acidosis. Our hypothesis is that glutamine may induce alternative metabolic routes when using PDK inhibitors. Glutamine is an important amino acid that can support cell proliferation³. Furthermore, long-term exposure of cancer cells to acidic pH leads to a metabolic reprogramming toward glutamine metabolism³. To measure metabolic adaptations of cancer cells to acidic environment and in response to DCA, we studied metabolic fluxes using ¹³C-NMR spectroscopy. This will provide a strong rationale for the association of treatments to be used in combination with PDK inhibitors.

Methods

Cancer cell lines with different metabolic profiles (SiHa; oxidative, HCT-116; glycolytic) and their pH-acidic adapted clones were incubated with +/- 5 mM of DCA and one ¹³C tracer (glucose-¹³C6 or glutamine-5-¹³C). After 24h, extracellular media were collected and polar cellular metabolites were extracted. ¹³C-NMR spectra of cellular extracts and media were acquired on a 600 MHz Bruker NMR equipped with a cryoprobe. The acquisition time was 0.8 s with 1024 repetitions and 10 s of interpulse delay. Spectrum analysis, assignment and quantification were performed with the Amix software. We also assessed, by proliferation assay, the toxicity of DCA in each cell lines and their acidic clones.

Results

DCA induced more toxicity in HCT-116 and SiHa acidic clones compared to the parental cell lines (fig.1). The production of intracellular lactate from glucose-¹³C6 in HCT-116 was 90± 2% lower in the acidic clones than the parental cells (fig.2). We detected no lactate in the SiHa acidic clones. DCA decreased the intracellular lactate production from glucose of 57± 7% in HCT-116 parental cells, of 83± 3% in HCT-116 acidic clones and of 54± 4% in SiHa parental cells (fig.2). Similar results were found for extracellular lactate. DCA inhibited the conversion of pyruvate to acetyl-CoA and CO2, this last being converted into bicarbonate, quantifiable in extracellular media spectra. Figure 3 shows that the production of bicarbonate from glucose-¹³C6 was lower in the acidic clones than in the parental cells (lower of 68± 23% for HCT-116 and 67± 20% for SiHa). DCA increased bicarbonate production of 17± 3% in HCT-116 parental cells and of 356%± 28% in their acidic clones (fig3). DCA had no significant effect on SiHa parental and acidic clones. DCA also decreased alanine production and pyruvate export in HCT-116 parental cell lines, these metabolites were not detectable in the other cell lines. Figure 4 shows that basal glutamine-5-¹³C consumption was higher in acidic clones than in parental cell lines (14± 3% for HCT-116 and 69± 29% for SiHa). In the presence of DCA, glutamine consumption was increased in HCT-116 parental and acidic cells and in SiHa parental and acidic cells (33± 8%, 18± 7%, 25±17% and 32± 9% respectively). Interestingly, DCA decreased glutamate production from glutamine in all cell lines and increased aspartate production from glutamine in both parental cell lines. Aspartate was not detectable in acidic clones.

Discussion

Although the toxicity of DCA was more important in acidic clones, they adapted their metabolism to be less dependent on glucose than parental cells (lower lactate and bicarbonate production) and more dependent of glutamine metabolism (more glutamine consumption). Metabolic modulation of DCA was dependent of the cell type and acidic environment. Glutamine consumption was increased in the presence of DCA suggesting a compensatory mechanism. Interestingly, in the presence of DCA, glutamate production was decreased but aspartate production was increased. These observations warrant further investigation to understand to role of glutamine pathway in the response of tumor cells to DCA treatment.

Conclusion

Using ¹³C-NMR spectroscopy allow us to study cancer cell metabolic adaptation following PDK inhibition and under acidosis.

Acknowledgements

No acknowledgement found.