Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells

Abstract

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.

Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.

Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Keywords: CE-TOFMS; Cisplatin resistance; Glutamine synthetase; Metabolome; Ovarian cancer.

Fig. 1

Increase in CDDP resistance and global metabolic changes in A2780cis cells. a Effects of CDDP treatment on the viability of A2780 and A2780cis cells. Cell viability was measured at 48 h after treatment using the MTT assay. b For colony formation assays, A2780 and A2780cis cells were cultured in the presence of CDDP (concentrations as indicated). Colonies were counted 9 days after plating. c Score plots of principal component analysis (PCA) of 189 intracellular metabolite levels in A2780 and A2780cis cells measured using CE-TOFMS. The contribution rate of PC1 and PC2 were 74.4 and 10.9%, respectively. d Volcano plots with the fold change of each metabolite and p values calculated using the Student’s t-test (p < 0.05). The averages metabolite levels in A2780cis cells were compared with those in A2780 cells (n = 3). Red dots depict significantly increased metabolites in A2780cis cells. Blue dots depict significantly decreased metabolites in A2780cis cells. Gray dots depict metabolites without significant differences. See also Table S2. e Levels of glutamine (Gln), glutamate (Glu), and glutathione (GSH) in A2780 and A2780cis cells. Data are shown as the mean ± SD of the three independent experiments. Statistical significance was determined using the Student’s t-test (**p < 0.01, ***p < 0.001)

Fig. 2

Metabolic flux analysis using isotopically labelled glutamine in A2780 and A2780cis cells. a, b, and c Isotopologue distribution of metabolites in A2780 and A2780cis cells. Cells were incubated with medium containing glutamine isotopically labeled at all five carbon atoms (¹³C₅-glutamine) for the indicated time periods. Carbon fluxes from glutamine to Glu, GSH, and α-ketoglutarate (α-KG) were determined using CE-TOFMS. Each bar color corresponds to the number of ¹³C replaced with ¹²C in the metabolites. Data are shown as the mean ± SD of three independent experiments. d A pathway map of glutamine metabolism. Metabolites and catalytic enzymes are shown in black and blue, respectively. The colored dots show the ¹³C isotopically labeled metabolites, and the color corresponds to the icons in Fig. 2a–c on the left

Fig. 3

Glutamine starvation reduces the GSH level and CDDP resistance in A2780cis cells. a Levels of Gln, Glu, and GSH in A2780 and A2780cis cells cultured in the presence (+) or absence (−) of glutamine. b Effects of glutamine starvation on CDDP resistance. A2780 and A2780cis cells were treated with 3 μM CDDP in the presence (+) or absence (−) of glutamine for 48 h. c A2780 and A2780cis cells were cultured for 48 h in medium containing CDDP (0 or 10 μM) and the GLS inhibitor compound 968 (0, 1, 3, or 10 μM). Cell viability was measured using the MTT assay. Data are shown as the mean ± SD of the three independent experiments. The differences were analyzed by using the Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

CDDP resistance in A2780cis cells is caused by DNA methylation-medicated silencing of GS expression. a RT-PCR analysis of glutamine metabolism enzymes. b Western blotting of glutamine metabolism enzymes. “+“indicates culture medium with normal glutamine concentration, and “−“indicates culture medium without glutamine. Cells were cultured in the absence or presence of glutamine for 48 h. c RT-PCR analysis of GS expression in A2780cis cells. Cells were cultured for 72 h in the absence or presence of 5-Aza-dC (2 μM). d Effects of 5-Aza-dC on CDDP-induced cytotoxicity. A2780cis cells were cultured for 72 h in medium containing CDDP (0 or 10 μM) and 5-Aza-dC (0, 1, 2, or 5 μM). Cell viability was measured using the MTT assay. e Confirmation of GS knockdown in A2780 cells using RT-PCR analysis. f Effects of CDDP knockdown on CDDP-induced cytotoxicity. A2780 cells transfected with control siRNA or GS siRNA were cultured for 72 h in the presence of CDDP (concentrations as indicated). Cell viability was measured using the MTT assay. Data are shown as the mean ± SD of the three independent experiments. The differences were analyzed using the Student’s t-test or one-way ANOVA with Dunnett’s multiple comparison (*p < 0.05, **p < 0.01)

Fig. 5

A model for CDDP resistance development via reprogramming of glutamine metabolism in ovarian cancer cells. In CDDP-sensitive cancer cells, both GLS and GS are expressed, and low levels of GSH are produced from glutamate. In contrast, in CDDP-resistant cells, GS expression is suppressed by DNA methylation, whereas GLS expression is maintained, and thereby high levels of GSH are produced. This reprogramming of glutamine metabolism causes CDDP resistance