The combination of orlistat, lonidamine and 6-diazo-5-oxo-L-norleucine induces a quiescent energetic phenotype and limits substrate flexibility in colon cancer cells

Abstract

Cancer upregulates glycolysis, glutaminolysis and lipogenesis, and induces a catabolic state in patients. The concurrent inhibition of both tumor anabolism and host catabolism, and the energetic consequences of such an approach, have not previously been fully investigated. In the present study, CT26.WT murine colon cancer cells were treated with the combination of anti-anabolic drugs orlistat, lonidamine and 6-diazo-5-oxo-L-norleucine (DON; OLD scheme), which are inhibitors of the de novo synthesis of fatty acids, glycolysis and glutaminolysis, respectively. In addition, the effects of OLD scheme sumplemented with the combination of anti-catabolic compounds, namely growth hormone, insulin and indomethacin (GII scheme), were also evaluated. The effects of the compounds used in combination on CT26.WT cell viability, clonogenicity and energetic metabolism were assessed in vitro. The results demonstrated that the anti-anabolic approach reduced cell viability, clonogenicity and cell cycle progression, and increased apoptosis. These effects were associated with decreased oxidative phosphorylation, glycolysis and fuel flexibility. Furthermore, the anti-catabolic scheme, alone or supplemented with anti-anabolic compounds, did not favor tumor growth. These findings indicated that the simultaneous pharmacological inhibition of tumor anabolism and host catabolism exhibits antitumor effects that should be further evaluated.

Keywords: colon cancer; de novo fatty acid synthesis; glutaminolysis; glycolysis; metabolism.

Figure 1.

The inhibition of the de novo synthesis of fatty acids, glycolysis and glutaminolysis diminishes cell viability and clonogenicity. (A) Percentage of cell viability after 72 h of treatment with each scheme. (B) Treated cells with either the OLD control (1), OLD (2), GII control (3), GII (4), 6 drugs control (5), or 6 drugs (6) conditions, after 72 h. (C) Percentage of colony formation 14 days after the 72 h treatment with each scheme. (D) CT26.WT plates from either the OLD control (1), OLD (2), GII control (3), GII (4), 6 drugs control (5), or 6 drugs (6) conditions, after 14 days. Data are expressed as mean ± SEM. Scale bars, 300 µm. ****P<0.0001. OLD, orlistat + lonidamine + DON; GII, growth hormone + insulin + indomethacin; 6 drugs, OLD + GII.

Figure 2.

The anti-anabolic drug combinations block cell cycle progression and stimulates apoptosis. (A) Percentage of cells in each phase of cell cycle after 72 h of treatment with each scheme. (B) ModFit diagrams showing cell cycle distribution from either the OLD control (1), OLD (2), GII control (3), GII (4), 6 drugs control (5), or 6 drugs (6) conditions. (C) Percentage of cells either alive, on early apoptosis, on late apoptosis, or on necrosis, after 72 h of treatment with each scheme. (D) Diva diagrams showing alive (Q1), necrotic (Q2), late apoptotic (Q3), or early apoptotic (Q4) cells, from either the OLD control (1), OLD (2), GII control (3), GII (4), 6 drugs control (5), or 6 drugs (6) conditions. (E and F) Western blot evaluation (E) and densitometric analysis (F) of cleaved caspase-3 among all the schemes. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. OLD, orlistat + lonidamine + DON; GII, growth hormone + insulin + indomethacin; 6 drugs, OLD + GII.

Figure 3.

Effects of the anti-anabolic drug schemes on the oxidative phosphorylation. (A and B) Mitochondrial respiration measured with the XF Cell Mito Stress Test. (C) Individual parameters for mitochondrial respiration. All the measurements were done 14 h after treatment. Data are expressed as mean ± SEM. OLD, Orlistat + lonidamine + DON; GII, growth hormone + insulin + indomethacin; 6 drugs, OLD + GII; OCR, oxygen consumption rate; ECAR, extracellular acidification rate.

Figure 4.

The anti-anabolic combinations limit the energetic production from glycolysis and modify the energetic phenotype. (A) Energetic production through glycolysis, measured with the XF Glycolysis Stress Test. (B) Individual parameters for glycolysis. (C) Energetic phenotype chart involving OCR and ECAR, under basal measurements and after the maximal stress induced by FCCP. All the measurements were done 14 h after treatment. Data are expressed as mean ± SEM. *P<0.05, and **P<0.01. OLD, orlistat + lonidamine + DON; GII, growth hormone + insulin + indomethacin; 6 drugs, OLD + GII; OCR, oxygen consumption rate; ECAR, extracellular acidification rate.

Figure 5.

The OLD scheme induces an increase in dependency towards glucose. (A and B) Cellular respiration corresponding to substrate dependency (A) and capacity (B) obtained with the XF Mito Fuel Flex Test. (C and D) Fuel oxidation diagrams representing flexibility and dependency towards the three energetic substrates with OLD control (C) or OLD treatment (D). The sum of flexibility and dependency indicates the capacity. Data are expressed as mean ± SEM. All the measurements were done 14 h after treatment. OLD, Orlistat + lonidamine + DON; GlucD, glucose dependency; LD, long-chain fatty acid dependency; GlutD, glutamine dependency; GlucC, glucose capacity; LC, long-chain fatty acid capacity; GlutC, glutamine capacity; OCR, oxygen consumption rate.