Increased drug resistance is associated with reduced glucose levels and an enhanced glycolysis phenotype

Abstract

Background and purpose: The testing of anticancer compounds in vitro is usually performed in hyperglycaemic cell cultures, although many tumours and their in vivo microenvironments are hypoglycaemic. Here, we have assessed, in cultures of tumour cells, the effects of reduced glucose levels on resistance to anticancer drugs and investigated the underlying cellular mechanisms.

Experimental approach: PIK3CA mutant (AGS, HGC27), and wild-type (MKN45, NUGC4) gastric cancer cells were cultured in high-glucose (HG, 25 mM) or low-glucose (LG, 5 mM) media and tested for sensitivity to two cytotoxic compounds, 5-fluorouracil (5-FU) and carboplatin, the PI3K/mTOR inhibitor, PI103 and the mTOR inhibitor, Ku-0063794.

Key results: All cells had increased resistance to 5-FU and carboplatin when cultured in LG compared with HG conditions despite having similar growth and cell cycle characteristics. On treatment with PI103 or Ku-0063794, only the PIK3CA mutant cells displayed increased resistance in LG conditions. The PIK3CA mutant LG cells had selectively increased p-mTOR, p-S6, p-4EBP1, GLUT1 and lactate production, and reduced reactive oxygen species, consistent with increased glycolysis. Combination analysis indicated PI103 and Ku-0063794 were synergistic in PIK3CA mutant LG cells only. Synergism was accompanied by reduced mTOR signalling and increased autophagy.

Conclusions and implications: Hypoglycaemia increased resistance to cytotoxic agents, especially in tumour cells with a high dependence on glycolysis. Dual inhibition of the PI3K/mTOR pathway may be able to attenuate such hypoglycaemia-associated resistance.

Keywords: PI3K; chemoresistance; glycolysis; hypoglycaemia; metabolism.

Figure 1

Effect of extracellular glucose concentrations on drug sensitivity. The IC₅₀ values of treatment with 5-FU, carboplatin, PI103 and Ku-0063794 for 72 h on PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells cultured in HG or LG are displayed. Data shown are the mean log IC₅₀ values ± SD from five independent experiments. *P < 0.05.

Figure 2

Effect of extracellular glucose concentrations on AMPK, autophagy and mTOR signalling. (A) Western blot analysis of phosphorylated AMPK, total AMPK, LC3B1 and LCB2 levels in PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells cultured in HG or LG conditions for 24 h. Data shown are representative of three independent experiments. GAPDH was used as loading control. (B) Levels of p-AKT, p-mTOR, p-S6 and p-4EBP1 in PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells cultured in HG or LG conditions at 24 h determined by elisa. Data shown are the mean (±SD) absorbance values of phosphorylated protein normalized to total protein from three independent experiments. *P < 0.05.

Figure 3

Effect of glucose concentrations on glucose transport, lactate production and apoptosis. (A) Western blot analysis of GLUT1 and MCT4 in PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells cultured in HG or LG at 24 h. Data shown are representative of three independent experiments. GAPDH was used as loading control. (B) Levels of extracellular lactate (ODU, optical density units; cells without DMEM were used as a control), ROS and apoptosis after PI103 and Ku-0063794 treatment in PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells cultured in HG or LG conditions for 24 h are displayed. Data shown are the mean (±SD) values from three independent experiments. *P < 0.05.

Figure 4

Effect of 24 h treatment with IC₅₀ concentrations of DMSO, PI103, Ku-0063794, PI103 with one-fifth IC₅₀ Ku-0063794 and Ku-0063794 with one-fifth IC₅₀ PI103 on mTOR signalling (p-AKT, p-mTOR, p-S6, p-4EBP1; measured by elisa), extracellular lactate (measured by colorimetric assay) and apoptosis (measured by elisa) in PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells. For analysis of protein expression, the mean (±SD) absorbance value of phosphorylated protein normalized to total protein was calculated. Data shown are the average fold difference (±SD) of values from cells cultured in LG compared with HG conditions, from three independent experiments.

Figure 5

Phase contrast images of PIK3CA mutant (MT; AGS, HGC27) and wild-type (WT; MKN45, NUGC4) GC cells at 24 h post-exposure to the combination of PI103 at IC₅₀ and Ku-0063794 at one-fifth IC₅₀, and the combination of Ku-0063794 at IC₅₀ and PI103 at one-fifth IC₅₀. Images were captured at 20× magnification and are representative of two independent experiments.

Figure 6

Model of ‘synthetic lethal addiction’ to rationalize selective synergy of PI103 and Ku-0063794 in PIK3CA mutant cells cultured in LG conditions. In PIK3CA wild-type cells, PI3K activates AKT, mTOR, S6, 4EBP1, protein translation and glycolysis (black arrows). In PIK3CA mutant cells, additional activation of AKT, mTOR, S6, 4EBP1, protein translation and glycolysis occurs (red arrows). Under glucose deprivation and in cells with enhanced glycolysis, GLUT1 levels are increased, leading to activation of mTOR signalling, protein translation and glycolysis (blue arrows). Synergy between PI103 and Ku-0063794 is observed only in PIK3CA mutant cells grown in LG conditions as these cells are dependent on hyperactivation of the PI3K/mTOR pathway. PIK3CA mutant cells cultured in HG conditions or PIK3CA wild-type cells in LG or HG conditions are less dependent on PI3K/mTOR activation, and hence the dual blockade of the PI3K/mTOR pathway is less detrimental. Autophagy occurs in cells with synergy due to the inhibition of mTOR.