Metabolic sensitivity of pancreatic tumour cell apoptosis to glycogen phosphorylase inhibitor treatment

Abstract

Inhibitors of glycogen breakdown regulate glucose homeostasis by limiting glucose production in diabetes. Here we demonstrate that restrained glycogen breakdown also inhibits cancer cell proliferation and induces apoptosis through limiting glucose oxidation, as well as nucleic acid and de novo fatty acid synthesis. Increasing doses (50-100 microM) of the glycogen phosphorylase inhibitor CP-320626 inhibited [1,2-(13)C(2)]glucose stable isotope substrate re-distribution among glycolysis, pentose and de novo fatty acid synthesis in MIA pancreatic adenocarcinoma cells. Limited oxidative pentose-phosphate synthesis, glucose contribution to acetyl CoA and de novo fatty acid synthesis closely correlated with decreased cell proliferation. The stable isotope-based dynamic metabolic profile of MIA cells indicated a significant dose-dependent decrease in macromolecule synthesis, which was detected at lower drug doses and before the appearance of apoptosis markers. Normal fibroblasts (CRL-1501) did not show morphological or metabolic signs of apoptosis likely due to their slow rate of growth and metabolic activity. This indicates that limiting carbon re-cycling and rapid substrate mobilisation from glycogen may be an effective and selective target site for new drug development in rapidly dividing cancer cells. In conclusion, pancreatic cancer cell growth arrest and death are closely associated with a characteristic decrease in glycogen breakdown and glucose carbon re-distribution towards RNA/DNA and fatty acids during CP-320626 treatment.

Figure 1

(A) Cell count before and after 72 h dose-escalating CP-320626 treatment of MIA pancreatic adenocarcinoma cells in culture. Cell numbers before the study and 72 h later in response to 50 μM CP-320626 treatment are not statistically different from the starting cell number. Control cells were grown for 72 h with vehicle only. (B) TUNEL assay indicates 4% apoptotic MIA cells formation in 50 μM CP-320626-treated cultures, which increases about seven-fold, to 28%, after 100 μM CP-320626 treatment. (C) Cell count before and after 72 h dose-escalating CP-320626 treatment of CRL-1501 human fibroblasts. (D) TUNEL assay indicates only 1–5% apoptotic CRL-1501 cell formation in 50 μM CP-320626-treated cultures. (n=6; mean+s.d.; ^*P<0.05, ^**P<0.01).

Figure 2

Optical density (OD) of MIA cultures after 72 h dose-escalating CP-320626 treatment in the MTS cell proliferation/survival assay. This test indicates significantly fewer MIA cells with deprived oxidative metabolism after increasing doses of CP-320626 treatment. This experiment was performed in comparison with 2-deoxy-D-glucose, which is a well-known growth-inhibitory substrate through cell cycle arrest and apoptosis (n=8; mean±s.d.; ^*P<0.05, ^**P<0.01).

Figure 3

SIDMAP of MIA cells after treatment with increasing doses of the glycogen phosphorylase inhibitor CP-320626 indicates severe changes in intracellular glucose carbon distribution. Carbon dioxide release from glucose is decreased, as demonstrated by a fall in ¹³C/¹²C ratios in panel A; the decrease in pentose cycle direct glucose oxidation and recycling into lactate is shown in panel B as per cent (%) of glycolysis; the decrease in nucleic acid (RNA) ribose ¹³C molar enrichment is shown in panel C as ¹³C ∑m_n, and the severe decrease in TCA cycle anabolic glucose use (anaplerosis) is shown in panel D as per cent (%) of glucose oxidation within the cycle (n=6; mean+s.d.; ^*P<0.05, ^**P<0.01).

Figure 4

Continued SIDMAP reflecting decreased fatty acid synthesis in MIA cells after treatment with increasing doses of the glycogen phosphorylase inhibitor CP-320626. In control cultures, about 18% of acetyl units and about 52% of intracellular palmitate arise from glucose, expressed as per cent (%) of the total. CP-320626 treatment decreased both acetyl unit synthesis and de novo fatty acid palmitate synthesis from glucose in a dose-dependent fashion (n=6; mean+s.d.; ^*P<0.05, ^**P<0.01).

Figure 5

Metabolic profile changes associated with glycogen phosphorylase inhibitor treatment in MIA pancreatic adenocarcinoma cells. MIA cells in the absence of CP-320626 utilise glucose as the major substrate for de novo nucleic acid and fatty acid synthesis as shown to the left (A). CP-3206262 limits glycogen breakdown as indicated on the panel to the right (B). As a result, tumour cells become less capable of synthesising nucleic acid ribose and fatty acids, which significantly limits their cycle progression and growth. One interesting feature of this metabolic profile is that it appears at lower concentrations of CP-320626 and precedes apoptosis formation. (G6P: glucose -6-P; G1P: glucose-1-P; F6P: fructose-6-P; GAP: glyceraldehyde-3-P; PYR: pyruvate; OAA: oxaloacetate).