Department of Surgery, Brigham and Women's Hospital, Boston, MA.
Department of Pathology, Brigham and Women's Hospital, Boston, MA.
Surgery. 2020 Jan;167(1):87-93. doi: 10.1016/j.surg.2019.06.042. Epub 2019 Sep 12.
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.
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.
Ketogenic diet plus N-acetylcysteine decreased in vivo tumor volume compared to standard diet (22.5 ± 12.4 mm vs 147 ± 54.4 mm, 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).
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.
间变性甲状腺癌是一种侵袭性和致命性的恶性肿瘤。许多晚期癌症的特征是葡萄糖依赖性，导致氧化应激和细胞增殖。因此，我们试图确定低葡萄糖环境（体外）或生酮饮食（体内）是否可以与抗氧化剂 N-乙酰半胱氨酸联合抑制间变性甲状腺癌肿瘤生长。
体内，将间变性甲状腺癌细胞系 8505C 注射到裸鼠中（每组 n = 6）。第 1 组喂食标准饮食；第 2 组喂食生酮饮食；第 3 组给予标准饮食加 N-乙酰半胱氨酸（饮用水中 40 mM）；第 4 组喂食生酮饮食加 N-乙酰半胱氨酸。测量肿瘤体积、酮体和葡萄糖。对肿瘤进行 H&E 染色和 Ki-67 和 Caspase 3 的免疫组化染色。体外，8505C 细胞在高葡萄糖（25 mM）、低葡萄糖（3 mM）、高葡萄糖加 N-乙酰半胱氨酸（200 μM）或低葡萄糖加 N-乙酰半胱氨酸中培养 96 小时。我们进行 CyQUANT 增殖（Thermo Fisher Scientific，Waltham，MA）、Agilent Seahorse 糖酵解应激（加利福尼亚州圣克拉拉）和活性氧化应激测定。
与标准饮食（22.5 ± 12.4 mm 比 147 ± 54.4 mm，P <.05）和标准饮食加 N-乙酰半胱氨酸（P <.05）相比，生酮饮食加 N-乙酰半胱氨酸降低了体内肿瘤体积。与标准饮食组相比，生酮饮食组的小鼠血液酮体水平明显升高（第 5 周时为 1.74 mmol/L 比 0.38 mmol/L，P <.001）。然而，生酮饮食组和标准饮食组的血糖水平没有明显差异。与高葡萄糖相比，低葡萄糖加 N-乙酰半胱氨酸培养的细胞增殖显著减少（98.1 ± 5.0 相对荧光单位比 157.8 ± 2.1 相对荧光单位，P <.001）。与高葡萄糖相比，N-乙酰半胱氨酸加入低葡萄糖会降低糖酵解功能（39.0 ± 2.2 mpH/min/细胞比 89.1 ± 13.2 mpH/min/细胞，P <.001）和高葡萄糖加 N-乙酰半胱氨酸（37.4 ± 2.5 mpH/min/细胞比 70.3 ± 3.3 mpH/min/细胞，P <.001）。与高葡萄糖相比，低葡萄糖加 N-乙酰半胱氨酸降低了活性氧化应激（119 ± 34.7 相对荧光单位比 277 ± 16.0 相对荧光单位，P =.014）。
生酮饮食或葡萄糖限制联合抗氧化剂 N-乙酰半胱氨酸显著减少了体内和体外的肿瘤生长。需要进一步研究来探索这些代谢疗法在间变性甲状腺癌中的应用。
