Inhibition of glycolysis disrupts cellular antioxidant defense and sensitizes HepG2 cells to doxorubicin treatment

Abstract

Increased glucose consumption is a known hallmark of cancer cells. Increased glycolysis provides ATP, reducing agents and substrates for macromolecular synthesis in intensely dividing cells. Therefore, inhibition of glycolysis is one strategy in anticancer therapy as well as in improved efficacy of conventional anticancer chemotherapeutic agents. One such agent is doxorubicin (DOX), but the mechanism of sensitization of tumor cells to DOX by inhibition of glycolysis has not been fully elucidated. As oxidative stress is an important phenomenon accompanying DOX action and antioxidant defense is closely related to energy metabolism, the aim of the study was the evaluation of oxidative stress markers and antioxidant abilities of cancer cells treated with DOX while glycolysis is inhibited. HepG2 cells were treated with DOX and one of three glycolysis inhibitors: 2-deoxyglucose, dichloroacetate or 3-promopyruvate. To evaluate the possible interaction mechanisms, we assessed mRNA expression of selected genes related to energy metabolism and antioxidant defense; oxidative stress markers; and reduced glutathione (GSH) and NADPH levels. Additionally, glutamine consumption was measured. It was demonstrated that the chemotherapeutic agent and glycolysis inhibitors induced oxidative stress and associated damage in HepG2 cells. However, simultaneous treatment with both agents resulted in even greater lipid peroxidation and a significant reduction in GSH and NADPH levels. Moreover, in the presence of the drug and an inhibitor, HepG2 cells had a reduced ability to take up glutamine. These results indicated that cells treated with DOX while glycolysis was inhibited had significantly reduced ability to produce NADPH and antioxidant defenses.

Keywords: 2-deoxyglucose; 3-promopyruvate; dichloroacetate; doxorubicin; glycolysis inhibitors; oxidative stress.

Figure 1

Relative HepG2 cell viability determined by MTT assay. The results were calculated as percentage of control cultures, which were averaged to define 100%. Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons on the basis of Tukey's HSD test were used.

Figure 2

Cell apoptosis/necrosis of HepG2 cells, stained with annexin V–FITC and propidium iodide for image cytometry. (A) Live, (B) early apoptotic, (C) late apoptotic and (D) necrotic cells. The results show one representative experiment of three independently performed that showed similar patterns.

Figure 3

Mean fluorescence intensity of CellRox probe presented as percentage of fluorescence in control cultures, which were averaged to define 100%. Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 4

Lipid peroxidation level in HepG2 cells on the basis of MDA and 4‐HAE concentration (μm). Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 5

AP site number per 100 kpb in HepG2 cells. Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 6

GSH level in HepG2 cells presented as percentage of control cultures, which were averaged to define 100%. Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 7

NADPH concentration in HepG2 cells (pmol·mg⁻¹ of protein). Values were presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 8

Glutamine consumption presented as percentage of control cultures and normalized to cell number. Values are presented as mean ± SD derived from three independent experiments. To compare more than two groups, one‐way ANOVA and post hoc multiple comparisons with Tukey's HSD test were used.

Figure 9

Summary of DOX inhibitory impact on tested genes’ expression. GLUT1, glucose transporter 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PCK2, phosphoenolpyruvate carboxykinase 2; PDK1, pyruvate dehydrogenase kinase 1; NNT, nicotinamide nucleotide transhydrogenase; SOD2, superoxide dismutase 2; GPX1, glutathione peroxidase 1.

Figure 10

NADPH cell sources influenced by DOX in conditions of glycolysis inhibition.