Metabolic Response of Pancreatic Carcinoma Cells under Treatment with Dichloroacetate

Abstract

In modern oncology, the analysis and evaluation of treatment response are still challenging. Hence, we used a ¹³C-guided approach to study the impacts of the small molecule dichloroacetate (DCA) upon the metabolic response of pancreatic cancer cells. Two different oncogenic PI3K-driven pancreatic cancer cell lines, 9580 and 10,158, respectively, were treated with 75 mM DCA for 18 h. In the presence of [U-¹³C₆]glucose, the effects of DCA treatment in the core carbon metabolism were analyzed in these cells using gas chromatography-mass spectrometry (GC/MS). ¹³C-enrichments and isotopologue profiles of key amino acids revealed considerable effects of the DCA treatment upon glucose metabolism. The DCA treatment of the two pancreatic cell lines resulted in a significantly decreased incorporation of [U-¹³C₆]glucose into the amino acids alanine, aspartate, glutamate, glycine, proline and serine in treated, but not in untreated, cancer cells. For both cell lines, the data indicated some activation of pyruvate dehydrogenase with increased carbon flux via the TCA cycle, but also massive inhibition of glycolytic flux and amino acid biosynthesis presumably by inhibition of the PI3K/Akt/mTORC axis. Together, it appears worthwhile to study the early treatment response in DCA-guided or accompanied cancer therapy in more detail, since it could open new avenues for improved diagnosis and therapeutic protocols of cancer.

Keywords: [U-13C6]glucose; dichloroacetate (DCA); isotopologue profiling; pancreatic cancer.

Figure 1

WST-1 viability/proliferation assays under treatment with DCA. Significant decrease in viability/proliferation in 9580 (a) and 10,158 cells (b) is shown at concentrations of 75 mM.

Figure 2

Typical core metabolism in cancer cells controlled by upregulated PI3K/Akt/mTORC. Green arrows indicate metabolic flux at high efficiencies. Notably, glucose uptake and utilization via glycolysis are upregulated. Flux via the TCA cycle and oxidative phosphorylation is downregulated by PDH inactivation due to phosphorylation catalyzed by PDK. When starting from [U-¹³C₆]glucose (M + 6) as a carbon substrate, the resulting ¹³C-isotopologues in key metabolites and amino acids (in blue boxes) are indicated. M + 3 and M + 2 indicate molecules carrying three or two ¹³C-atoms, respectively. PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase, ETC, electron transfer chain; HK2, hexokinase 2; ACLY, ATP-citrate lyase; IDH1, isocitrate dehydrogenase 1.

Figure 3

Overall ¹³C enrichment in analyzed amino acids of untreated control cells and DCA treated cells. Results are shown for 9580 (a) and 10,158 cells (b). DCA treatment of 9580 and 10,158 cancer cell lines led to a significant decrease in ¹³C-enrichment in alanine (Ala), aspartate (Asp), glutamate (Glu), glycine (Gly), proline (Pro) and serine (ser). * p < 0.05, ** p < 0.01 *** p < 0.0001 compared to controls. Mass fragments of silylated amino acids used for ¹³C-determination are indicated.

Figure 4

¹³C isotopologue profiles of serine, glycine, alanine, glutamate, proline and aspartate following incubation of 9580 cells with DCA compared to untreated controls. Relative fractions of isotopologues (M + 1–M + 5) in the overall ¹³C-labelled amino acids are indicated by different colors. ALT: alanine aminotransferase; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; SHMT: serine hydroxymethyltransferase; N = 3.

Figure 5

¹³C isotopologue profiles of serine, glycine, alanine, glutamate, proline and aspartate following incubation of 10,158 cells with DCA compared to untreated controls. Relative fractions of isotopologues (M + 1–M + 5) in the overall ¹³C-labelled amino acids are indicated by different colors. ALT: alanine aminotransferase; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; SHMT: serine hydroxymethyltransferase; N = 3.

Figure 6

Model for DCA-induced effects upon the core metabolism in 9580 and 10,158 cancer cells. Green arrows and symbols indicate increased metabolic activities and stimulatory effects. Red arrows and symbols indicate reduced metabolic activities and inhibitory effects. Notably, glucose utilization via glycolysis could be reduced due to the deactivation of PI3K/Akt. Flux via the TCA cycle and oxidative phosphorylation could be increased by PDH activation due to inhibition and deactivation of PDK. When starting from [U-¹³C₆]glucose (M + 6) as a carbon substrate, effects on the relative fractions of ¹³C-isotopologues in key metabolites and amino acids (in blue boxes) are indicated in red when reduced by DCA or in green when increased by DCA. M + 3, M + 2 and M + 1 indicate molecules carrying three, two or one ¹³C-atoms, respectively. PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase, ETC, electron transfer chain; HK2, hexokinase 2; ACLY, ATP-citrate lyase; IDH1, isocitrate dehydrogenase 1.