Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

Introduction
Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.
Methods
Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).
Results
Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.
Conclusions
Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.

引言
神经母细胞瘤（Neuroblastoma）是一种起源于神经嵴细胞的儿童恶性肿瘤，其典型特征为线粒体氧化磷酸化（mitochondrial oxidative phosphorylation）普遍下调。本研究旨在探究热量限制（calorie restriction）与生酮饮食（ketogenic diet）对神经母细胞瘤生长的干预效应，并监测该肿瘤氧化磷酸化系统潜在的适应性机制。
方法
通过向CD-1裸小鼠皮下注射两种具有不同遗传特征与治疗敏感性的神经母细胞瘤细胞系[SH-SY5Y与SK-N-BE(2)]，建立异种移植瘤（xenografts）模型。将小鼠随机分为4组，分别饲喂标准饲料、热量限制型标准饲料、长链脂肪酸型生酮饮食以及热量限制型生酮饮食。监测小鼠的肿瘤生长、生存期、代谢指标与体重变化。对肿瘤组织开展检测，以分析饮食干预诱导的增殖指数及多项氧化磷酸化系统相关参数的变化，包括呼吸链酶活性、蛋白质印迹分析（western blot analysis）、免疫组织化学（immunohistochemistry）检测以及线粒体DNA（mitochondrial DNA）含量测定。
结果
生酮饮食联合或不联合热量限制，均可显著抑制异种移植瘤模型的肿瘤生长，并延长小鼠生存期。神经母细胞瘤的生长抑制效应与血糖浓度降低显著相关，且在肿瘤生长受抑的饮食干预组中，Ki-67与磷酸化组蛋白H3（phospho-histone H3）的表达水平显著下调。与人类肿瘤组织一致，该神经母细胞瘤异种移植瘤模型呈现出显著降低的线粒体复合物II（mitochondrial complex II）活性，同时伴随线粒体氧化磷酸化整体水平低下，验证了该模型的可靠性。此外，神经母细胞瘤无法通过调控线粒体氧化磷酸化活性，以适应饮食干预所引发的营养供给改变。
结论
本研究数据显示，靶向神经母细胞瘤的代谢特征可为现有标准治疗方案提供新的研究维度。因此，我们建议将生酮饮食联合或不联合热量限制作为神经母细胞瘤患者的潜在辅助治疗方案，开展后续评估与研究。