In the present study, we used a bioluminescent mouse model of malignant glioma to demonstrate that a KD can: (1) significantly retard tumor growth; (2) prevent increases in ROS associated with tumor growth; and (3) shift overall gene expression in tumor tissue to a pattern seen in normal brain. When compared to the expression profile in normal brain, the KD exerts differential effects in brain tumor tissue, and appears to influence specific genes involved in regulation of ROS levels. Taken together, our data suggest that the underlying mechanisms likely involve complex alterations in cellular metabolism beyond a simple reduction in blood glucose, as previously hypothesized [21
The KD is a high-fat, low carbohydrate diet that has been successfully used to treat medically refractory epilepsy for many decades, particularly in children [23
]. Intriguingly, recent studies have highlighted potential uses for other neurological disorders [21
]. With respect to brain tumors, Seyfried and co-workers [2
] demonstrated that the KD or caloric restriction could extend survival in a mouse model of astrocytoma, and proposed that glucose restriction may be the critical factor, despite a multiplicity of other potential mechanisms [2
]. Specifically, while normal brain cells may readily adapt to using ketone bodies as an alternative source of energy, tumor cells are less metabolically flexible. There are myriad reasons for this difference, and changes in gene expression may affect other aspects of glycolysis, respiration and mitochondrial function [21
To explore other mechanisms, we used a bioluminescent GL261/C57BL/6 mouse model system in which the KD extends survival in a manner similar to that seen in the CT-A model system used by Seyfried [30
]. Serial in vivo
imaging of these tumors demonstrated that the KD slowed the overall rate of growth of these tumors, rather than increasing survival by selecting for a subpopulation of cells less influenced by the dietary change. To gain further insight into this observation, we undertook gene expression studies, and compared the profiles to previous studies involving KD treatment in normal rodent brain.
Noh et al [36
] studied the hippocampus of normal juvenile mice using a Rat Atlas 1.2 Array II cDNA expression array (Clontech Laboratories) containing 1176 genes. They found 42 genes that were differentially expressed after KD treatment, and interestingly, most encoded proteins are ordinarily involved in mitochondrial metabolic and intracellular signal transduction pathways. Similarly, Bough et al [37
] analyzed the effect of a calorie-restricted KD versus SD fed ad libitum
on gene expression in the hippocampi of normal male rats. They reported up-regulation of genes encoding elements of oxidative phosphorylation and other mitochondrial proteins, as well as some involved in mitochondrial biogenesis. Yet other investigators have studied the expression of genes involved in metabolism using various diets [38
], anti-diabetic drugs [39
] and genetically altered mice [40
While there is a dearth of information regarding the mechanisms underlying the putative anti-neoplastic effects of the KD, a number of studies have provided insights into the neuroprotective properties of the diet, and in particular, the role that ROS plays [7
]. This observation was of interest to us since ROS are known to be effector molecules involved in numerous intracellular pathways, including those regulating cellular autophagic/apoptotic responses to genotoxic stress, hypoxia and nutrient deprivation [9
]. Additionally, increased levels of ROS [8
] can lead to induction of angiogenesis and tumor growth through regulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) [9
]. Several signal transduction cascades activated by tyrosine kinase receptors act in part through ROS-dependent mechanisms [8
], and Akt activation by ROS may support tumor cell survival under hypoxic conditions [47
]. Finally, ROS may even contribute to the heterogeneity seen in brain tumors because of its differential effects in normoxic vs. hypoxic areas of a tumor [8
We found that our GL261 cells did indeed exhibit high ROS levels, and when maintained in vitro, responded with a significant reduction in ROS upon addition of ketones (Figure ). Consistently, in vivo imaging demonstrated increased ROS in tumor tissue from animals fed a SD (Figure ) and ex vivo analysis demonstrated the significant reduction of ROS in tumor tissue from animals fed a KD (Figure ). While the majority of the tumor showed ROS levels consistent with normal brain in animals fed a KD, it was of interest that a few cells appeared to maintain higher levels of ROS. This may be a reflection of the heterogeneity seen in these tumors. However, it is unlikely that these cells are clinically relevant because no increases in ROS-positive cells were observed over time. Thus, despite the presence of these cells, the many functions of ROS suggest possible pathways through which the KD may affect tumor growth other than alterations in glucose availability.
Given the pivotal role that ROS seemed to play in tumors as a response to the KD, we focused our analysis of gene expression patterns on specific genes involved in modulation of oxidative stress and antioxidant defense pathways. Our statistical analysis revealed that the KD did not substantially alter gene expression in normal mouse brain; however, overall expression in tumor tissue was affected such that it resembled that seen in normal brain issue (Figure ). Moreover, genes that were over-expressed in tumor relative to non-tumor tissue in animals fed a SD were under-expressed in tumor relative to normal tissue in animals fed a KD (Figure ). When we independently analyzed the effect of diet on tumor vs. normal tissue, it was evident that non-tumor tissue generally had a much more robust response to diet than did tumor tissue (Figure ).
One prominent gene in our analyses is Ptgs2
). We found that Cox2 expression was reduced to non-tumor levels when animals are fed a KD (Figure ). This was particularly intriguing as Cox2
inhibition is being explored as a treatment strategy for brain tumors [48
]. Inhibition of Cox2
has been correlated with increased apoptosis in some systems and has been associated with decreased endothelial cell spreading, migration and angiogenesis [48
]. These data are consistent with a previous report that the KD increased apoptosis and inhibited angiogenesis [30
Other genes involved in oxidative stress responses were also affected by the KD, including glutathione peroxidase 7
) and peroxiredoxin 4
), both of which play cellular protective roles [53
], and are similarly regulated as Cox2 - i.e., showing higher expression in tumor vs. normal tissue when animals are fed a KD, but not when they are fed a SD. Ziegler et al [55
] reported an increase in the activity of the Gpx enzyme in the hippocampus of normal rats fed a KD, but not in the cortex of cerebellum. This study did not examine gene expression profiles, nor did it separate out the isoforms of the Gpx enzyme.
In contrast to Gpx7
) - which also protects cells against oxidative stress [56
] - was more highly expressed in tumor vs. non-tumor tissue in animals fed a SD, as well as in normal brain when mice were fed a KD. Cytoglobin
also encodes a tumor suppressor protein [57
], and this may help explain why its expression is higher in tumor tissue from animals fed SD, but lower in tumor tissue from animals fed a KD.
Yet another potentially significant player is Nox organizing protein 1
) which is required for ROS generation by Nox1, along with the NOX activating protein 1
]. It is not clear why the expression of this gene is higher in tumor than in normal brain in animals fed KD, and why SD-fed animals show higher expression in non-tumor tissue. Adding further complexity is the protein encoded by cytochrome b245
, alpha polypeptide
), which directly interacts with both NOXO1 and NOX1, which in turn leads to the production of H2
. The expression of Cyba
changes with diet and tissue type in a manner identical to the gene encoding NOXO1. Thus, there are a number of genes involved in the formation of ROS through the Nox system, and we cannot rule out the possibility that the KD is less effective at reducing the production of ROS through this mechanism.
Finally, animals fed a KD showed higher expression the gene encoding the serine (or cysteine) peptidase inhibitor, clade B, member 1b (Serpinb1b
), a putative antioxidant enzyme [59
]. This was particularly evident in non-tumor tissue when compared to that seen in animals fed SD; however, Serpinb1b
expression was higher in KD-fed normal mice, whereas in the tumor groups, animals fed a SD exhibited higher levels. The reason for this difference is not yet clear, but at present, very little is known about the function of this gene product.