A75 TARGETING THE WARBURG EFFECT IN HEPATOCELLULAR CARCINOMA CELLS

Abstract

Background

The avid consumption of glucose (Glu) with concomitant lactate production by malignant cells is called the Warburg effect (WE). As most invasive tumours harbour this feature, this has proven of great clinical importance in detecting malignancies with PET scans. Unfortunately, in the liver, the detection of tumors is often compromised by the strong intrinsic Glu metabolic activity. Understanding the manner by which hepatocellular carcinoma (HCC) cells use Glu to proliferate is essential if we want to bypass the aforementionned difficulty.

Aims

Recently, targeting molecular mechanisms involved in the WE such as lactate dehydrogenase (LDH) has been advocated as a very promising anti-cancer target. We hypothesized that the modulation of LDH which catalyzes the conversion of pyruvate to lactate might have an impact on HCC cell tumorigenicity, since the production of lactate has been shown to increase tumor invasion.

Methods

We generated a mouse hepatoma cell line Dt81Hepa1-6 (Dt) derived from Hepa1-6 (H1-6) cells that displays greater tumorigenicity in vivo. We hypothesized that this increased tumorigenicity involved the WE. Cells were cultured in 25mM Glu over a period of 24-48h. Glu uptake was assessed using a fluorescent Glu analog, 2-NBDG. mRNA and metabolite quantification were respectively measured by qPCR and HPLC. Aerobic glycolysis was inhibited using increasing doses of sodium oxamate (SODOX), a classic inhibitor of LDH, to reduce lactate production. LDH activity was measured by the Biochemical core facility of CRCHUM. MTT assay was used to test the dose-response effects of SODOX on cell viability. Cell Doubling Time (CDT) was evaluated every 24h for 72h with SODOX (100 mM).

Results

Dt showed an increased ability to uptake Glu in low extracellular Glu in comparison with H1-6 (3 ± 0.3 vs 1.4 ± 0.1DO/μg prot, P<0.01). Increasing the extracellular Glu concentration led to an increase in Glu uptake only in Dt cells (P<0.001). qPCR and metabolite analysis showed that Dt displayed a higher glycolytic rate than H1-6, with greater ATP production (P<0.001). We then investigated the effect of SODOX on these cells. First, LDH acticvity was reduced in a dose-response manner after 24h with SODOX (100mM, P<0.01). MTT revealed that SODOX dose-dependently decreased cell viability of both H1-6 and Dt cells but that Dt were significantly more resistant at 100mM SODOX (43 ± 1.5 vs 55 ± 2.6, P<0.01). Finally, CDT revealed that SODOX significantly slowed Dt proliferation; this effect was even more pronounced on H1-6 (P<0.001).

Conclusions

These results suggest that the WE is effective in HCC cells and that increased tumorigenesis is associated with metabolic changes in glucose metabolism that favor ATP production. The WE represents a potentially remarkable new target to treat HCC. Our preliminary results suggest that decreasing LDH activity is one of the ways to achieve this goal.

Funding Agencies

CHAIRE NOVARTIS DE LA FONDATION CANADIENNE DU FOIE DE L’UNIVERSITE DE MONTREAL