Ketogenic diet combined with antioxidant N-acetylcysteine inhibits tumor growth in a mouse model of anaplastic thyroid cancer

Background: Anaplastic thyroid cancer is an aggressive and fatal malignancy. Many advanced cancers are characterized by glucose dependency, leading to oxidative stress and cellular proliferation. Therefore, we sought to determine if a low glucose environment (in vitro) or a ketogenic diet (in vivo) could inhibit anaplastic thyroid cancer tumor growth when combined with the antioxidant N-acetylcysteine. Methods: In vivo, nude mice were injected with the anaplastic thyroid cancer cell line 8505C (n = 6/group). Group 1 was fed a standard diet; Group 2 was fed a ketogenic diet; Group 3 was given standard diet with N-acetylcysteine (40 mM in the drinking water); and Group 4 was fed ketogenic diet with N-acetylcysteine. Tumor volumes, ketones, and glucose were measured. H&E stains and immunohistochemistry for Ki-67 and Caspase 3 were performed on the tumors. In vitro, 8505C cells were cultured in high glucose (25 mM), low glucose (3 mM), high glucose plus N-acetylcysteine (200 uM), or low glucose plus N-acetylcysteine for 96 hours. We performed CyQUANT proliferation (Thermo Fisher Scientific, Waltham, MA), Seahorse glycolytic stress (Agilent, Santa Clara, CA), and reactive oxidative stress assays. Results: Ketogenic diet plus N-acetylcysteine decreased in vivo tumor volume compared to standard diet (22.5 +/- 12.4 mm(3) vs 147 +/- 54.4 mm(3), P < .05) and standard diet plus N-acetylcysteine (P < .05). Blood ketone levels were significantly higher for the mice in the ketogenic diet group compared to standard diet (1.74 mmol/L vs 0.38 mmol/L at week 5, P < .001). However, blood glucose levels were not significantly different between ketogenic diet and standard diet groups. Cells cultured in low glucose plus N-acetylcysteine had significantly reduced proliferation compared to high glucose (98.1 +/- 5.0 relative fluorescence units vs 157.8 +/- 2.1 relative fluorescence units, P < .001). Addition of N-acetylcysteine to low glucose lowered glycolysis function compared to high glucose (39.0 +/- 2.2 mpH/min/cell vs 89.1 +/- 13.2 mpH/min/cell, P < .001) and high glucose plus N-acetylcysteine (37.4 +/- 2.5 mpH/min/cell vs 70.3 +/- 3.3 mpH/min/cell, P < .001). Low glucose plus N-acetylcysteine decreased reactive oxidative stress compared to high glucose (119 +/- 34.7 relative fluorescence units vs 277 +/- 16.0 relative fluorescence units, P = .014). Conclusion: The combination of a ketogenic diet or glucose restriction with the antioxidant-N-acetylcysteine significantly reduced tumor growth in vivo and in vitro. Further studies are warranted to explore these metabolic therapies in anaplastic thyroid cancer. (C) 2019 Elsevier Inc. All rights reserved.