Pharmacological Vitamin C Treatment Impedes the Growth of Endogenous Glutamine-Dependent Cancers by Targeting Glutamine Synthetase

Abstract

Purpose: Glutamine synthetase (GS) is the only currently known enzyme responsible for synthesizing endogenous glutamine (Gln). GS exerts a critical role in the oncogenesis of endogenous Gln-dependent cancers, making it an attractive target for anti-tumor therapies. A mixed-function oxidation system consisting of vitamin C (VC), oxygen, and trace metals can oxidize GS and promote its degradation. The current study aims to explore the effect of pharmacological VC treatment on GS. Methods: Endogenous Gln-dependent cancer lines (breast cancer MCF7 and prostate cancer PC3) were selected to establish chronic Gln-deprived MCF7 and PC3 cell models. The expression of GS in parental and chronic Gln-deprived tumor cells exposed to VC treatment and control was determined by Western blot analysis. The anti-cancer effects of VC on parental and chronic Gln-deprived tumor cells were assessed by CCK-8 and annexin V-FITC/PI FACS assays. In addition, changes in cellular reactive oxygen species (ROS), glutathione (GSH) levels and NADPH/NADP + ratio were analyzed to explore the underlying mechanisms. Moreover, BALB/c nude mice xenografting with parental and chronic Gln-deprived prostate cancer cells were constructed to evaluate the in vivo therapeutic effect of VC. Finally, tumor 13N-ammonia uptake in mice bearing prostate cancer xenografts was analyzed following treatment with VC and the expression of GS in xenografts were detected by immunohistochemistry. Results: Cells overexpressing GS were obtained by chronic Gln deprivation. We found that the cytotoxic effect of VC on cancer cells was positively correlated with the expression of GS. Additionally, VC treatment led to a significant increase in ROS production, as well as GSH depletion and NADPH/NADP + reduction. These changes could be reversed by the antioxidant N-acetyl-L-cysteine (NAC). Furthermore, pharmacological VC treatment exhibited a more significant therapeutic effect on xenografts of prostate cancer cells overexpressing GS, that could be well monitored by 13N-ammonia PET/CT imaging. Conclusion: Our findings indicate that VC can kill cancer cells by targeting glutamine synthetase to induce oxidative stress. VC could be used as an anti-cancer treatment for endogenous glutamine-dependent cancers.

Keywords: 13N-ammonia PET/CT; endogenous glutamine-dependent cancer; glutamine synthetase; redox stress; vitamin C.

FIGURE 1

Chronic Gln deprivation augments the levels of GS in MCF7 and PC3 cells. (A) GS protein in cancer cells (MCF7、PC3、0.06MCF7、0.06PC3) analyzed by Western blotting. (B) Relative GS protein contents were evaluated with β-tubulin as a loading control. All data are presented as means ± SEM, *p < 0.05, **p < 0.01, n = 3. GS, glutamine synthetase.

GRAPHICAL ABSTRACT

In normal condition, the endogenous Gln-dependent cancer cells generate Gln depending on glutamine synthetase (GS) for survival due to the limited ability of extracting circulating glutamine (Gln). Inhibition of GS by vitamin C (VC) limits Gln synthesis leads to a rapid depletion of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) as well as an accumulation of reactive oxygen species (ROS), ultimately induces cell death.

FIGURE 2

The cytotoxicity of VC increases in cancer cells overexpressed GS. (A) Cell viability evaluated by CCK-8 assay after treatment with different doses of VC for 6 h. (B) Cell viability evaluated by CCK-8 assay after treatment with VC (4 mM) for the indicated times. (C) IC50 of VC in cells evaluated by CCK-8 assay for the indicated times. (D) Cell apoptosis evaluated by annexin-V/PI assay after treatment with different doses of VC for 16 h. All data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3. VC, Vitamin C.

FIGURE 3

Cancer cells overexpressing GS show augmented protein degradation under VC treatment, which is reversed by NAC. (A) GS protein in cancer cells (MCF7、PC3、0.06MCF7、0.06PC3) analyzed by Western blotting under treatment with VC(4 mM), NAC(5 mM), VC(4 mM) + NAC(5 mM) for indicated times. (B) The relative GS contents of cancer cells (MCF7、0.06MCF7、PC3、0.06PC3) under treatment with VC(4 mM) for indicated times were calculated with β-tubulin as a loading control. (C) The relative expression of GS in cancer cells (MCF7、0.06MCF7、PC3、0.06PC3) under treatment with VC(4 mM), NAC(5 mM), VC(4 mM) + NAC(5 mM) for 3 h. All data are presented as means ± SEM, n = 3. VC, VitaminC; GS, glutaminesynthetase; NAC, N-acetyl-L-cysteine.

FIGURE 4

Cancer cells overexpressing GS show augmented redox imbalance under VC treatment. (A) Quantitative bar graphs of celllular ROS levels before and after treatment with VC (8mM, 3 h). (B) Quantitative bar graphs of celllular GSH levels before and after treatment with VC (8mM, 3 h). (C) Quantitative bar graphs of celllular NADPH/NADP + ratio before and after treatment with VC (8mM, 3 h). All data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3. VC, Vitamin C.

FIGURE 5

The cytotoxicity of VC reversed by NAC. Cell apoptosis evaluated by annexin-V/PI assay after treatment with VC(4 mM), NAC(5 mM), VC(4 mM) + NAC(5 mM) for indicated times. A and B. Pretreatment with 5 mM NAC for 1 h significantly prevented the cytotoxic effect induced by VC treatment. All data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3. VC, Vitamin C; NAC, N-acetyl-L-cysteine.

FIGURE 6

Xenografts of cells overexpressing GS show slow tumor growth under VC treatment. BALB/c nude mice (4–5 weeks old/male) were subcutaneously (s.c.) injected with 2 × 10⁶ PC3 cells. When the tumor sizes reached 50–80 mm³, ascorbate sodium (4 g/kg, twice a day) were intraperitoneally (i.p.) administered to the mice. After 16 days, all mice were euthanized and the tumors were analyzed. (A) Tumor volume and weight changed after treatment with VC. (B) Immunohistochemical analyses showed the expression of Ki-67 (proliferation marker) decreased in the VC treatment groups, with more obvious change in the 0.06PC3 group. All data are presented as means ± SEM, *p < 0.05, **p < 0.01, n = 3. VC, Vitamin C.

FIGURE 7

Functional 13N-ammonia PET/CT imaging of PC3 xenografts in vivo shows a significant reduction in 13N-ammonia uptake under VC treatment. (A) Tumor 13N-ammonia uptake in treated and control mice. 13N-ammonia PET/CT were conducted before (day 0) and 3 days after therapy initiation. Right panels, representative PET/CT scans showing change of tumor tracer uptake (red arrows). (B) Tumor-to-background ratio (TBR) was calculated and served as an indicator of tracer uptake. Mean TBR of 13N-ammonia PET/CT was reduced in VC treated mice compared to controls. Immunohistochemical analyses showed the expression of GS decreased in the VC treatment specimens. All data are presented as means ± SEM, *p < 0.05, n = 3. VC, Vitamin C; GS, glutamine synthetase.