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. 2015 Aug;22(4):577-91.
doi: 10.1530/ERC-15-0192. Epub 2015 Jun 4.

Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway

Lingqin Yuan  1 , Xiugui Sheng  2 , Adam K Willson  2 , Dario R Roque  2 , Jessica E Stine  2 , Hui Guo  1 , Hannah M Jones  2 , Chunxiao Zhou  3 , Victoria L Bae-Jump  3
Affiliations

Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway

Lingqin Yuan et al. Endocr Relat Cancer. 2015 Aug.

Abstract

Glutamine is one of the main nutrients used by tumor cells for biosynthesis. Therefore, targeted inhibition of glutamine metabolism may have anti-tumorigenic implications. In the present study, we aimed to evaluate the effects of glutamine on ovarian cancer cell growth. Three ovarian cancer cell lines, HEY, SKOV3, and IGROV-1, were assayed for glutamine dependence by analyzing cytotoxicity, cell cycle progression, apoptosis, cell stress, and glucose/glutamine metabolism. Our results revealed that administration of glutamine increased cell proliferation in all three ovarian cancer cell lines in a dose dependent manner. Depletion of glutamine induced reactive oxygen species and expression of endoplasmic reticulum stress proteins. In addition, glutamine increased the activity of glutaminase (GLS) and glutamate dehydrogenase (GDH) by modulating the mTOR/S6 and MAPK pathways. Inhibition of mTOR activity by rapamycin or blocking S6 expression by siRNA inhibited GDH and GLS activity, leading to a decrease in glutamine-induced cell proliferation. These studies suggest that targeting glutamine metabolism may be a promising therapeutic strategy in the treatment of ovarian cancer.

Keywords: glutamate dehydrogenase; glutaminase; mTOR/S6; ovarian cancer; siRNA.

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Figures

Figure 1
Figure 1
Glutamine metabolism promotes optimal cell proliferation. Ovarian cancer cells lines HEY (A), SKOV3 (B), and IGROV-1 (C) were treated in glutamine-free media supplemented with various concentrations of glutamine (0, 0.5, 2.0, 5.0, and 10.0 mM) for 48 h. Cell proliferation was assessed by MTT assay. The changes in glutaminase (GLS) expression upon the effects of glutamine were assessed using western blot. HEY, SKOV3, and IGROV-1 cells were treated with glutamine for 24 h. Treatment of glutamine reduced the expression of GLS in a dose-dependent manner (D). GLS changes in IGROV-1 cells after treatment with glutamine and glutamine starvation: glutamine starvation consistently increased the expression of GLS with the extension of starvation, and glutamine reduced the expression of GLS with the presence of glutamine for 48 h after starvation in IGROV-1 cells (E). GDH activity was assayed using a GDH assay kit in IGROV-1 cells. Glutamine greatly increased the activity of GDH (F). Data is shown as mean±s.e.m. of two trials (*P<0.05).
Figure 2
Figure 2
Glutamine affects cell cycle progression. HEY (A), SKOV3 (B), and IGROV-1 (C) cells were treated in glutamine-free media supplemented with various concentrations of glutamine (0, 0.5, 2.0, and 5.0 mM) for 48 h. Cell cycle analysis was performed using Cellometer. Depletion of glutamine induced cell cycle G1 phase arrest in ovarian cancer cells. The effects of glutamine on cyclin D1, CDK4, and p21 were examined by western blotting in HEY (D), SKOV3 (E), and IGROV-1 (F) cells after exposure to glutamine for 24 h at the indicated concentrations.
Figure 3
Figure 3
Depletion of glutamine induces cell apoptosis. HEY (A), SKOV3 (B), and IGROV-1 (C) cells were cultured with different concentrations of glutamine for 24 h. The apoptosis were detected using an Annexin-V FITC Kit. Depletion of glutamine induced significant cell apoptosis in the three cell lines. Data is shown as mean±s.e.m. of triplicates (*P<0.05).
Figure 4
Figure 4
Depletion of glutamine induces cell stress. The three cell lines were treated with glutamine for 24 h. The production of intracellular reactive oxygen species (ROS) was detected using DCFH-DA. Depletion of glutamine greatly increased ROS production (A). The expression of stress proteins in cells was detected using western blotting. Glutamine reduced the expression of stress proteins in HEY (B), SKOV3 (C), and IGROV-1 (D). *P<0.05.
Figure 5
Figure 5
Glutamine complements ATP production. The cells were cultured in media with various concentrations of glutamine for 24 h. The levels of cellular glucose uptake (A), cellular ATP production (B), and lactate in the media (C) were detected. The cells were treated with compound 968 or 3-BP for 24 h in 2.0 mM glutamine or glutamine-free media respectively. The levels of ATP (D) and lactate (E) were detected by ELISA assay. The expression of glycolytic proteins was detected by western blotting in HEY (F), SKOV3 (G), and IGROV-1 (H) after treatment with glutamine for 24 h. *P<0.05.
Figure 6
Figure 6
Glutamine activates the MAPK and mTOR/S6 pathways. The expression of phosphorylation of p-42/44 and p-S6 in HEY (A), SKOV3 (B), and IGROV-1 (C) cells was detected by western blotting after treatment with glutamine for 24 h. Glutamine increased protein expression of p-42/44 and p-S6 in the ovarian cancer cells (D and E).
Figure 7
Figure 7
Rapamycin inhibits glutaminolysis in ovarian cancer cells. HEY (A), SKOV3 (B), and IGROV-1 (C) cells were treated with various concentrations of rapamycin as indicated in their regular media for 24 h. Treatment with rapamycin reduced the expression of GLS and phosphorylation of p-S6. Rapamycin inhibited GDH activity in HEY cells (D) and blocked the cell proliferation induced by glutamine in the ovarian cancer cells after 60 h treatment (E). Western blotting showed rapamycin inhibited the expression of phosphorylation of p-S6 and GLS induced by glutamine in HEY (F), SKOV3 (G), and IGROV-1 (H) cells after 24 h treatment. *P<0.05.
Figure 8
Figure 8
Knockdown S6 by siRNA transfection reduces the expression of GLS and activity of GDH. HEY cells were transfected with RPS6 siRNA for 24 h. Western blotting showed the expression of phosphorylation of S6 was inhibited after siRNA transfection (A). HEY cells were treated with 2.0 mM glutamine for 24 h after siRNA transfection. Both of S6 siRNA and rapamycin reduced the expression levels of GLS and phosphorylation of S6 (B). S6 siRNA transfection decreased GDH activity induced by glutamine (C). HEY cells were treated with 2.0 mM glutamine for 48 h after siRNA transfection. Cell proliferation was determined by MTT assay (D). *P<0.05.
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