The ketogenic diet reverses gene expression patterns and reduces reactive oxygen species levels when used as an adjuvant therapy for glioma

Abstract

Background: Malignant brain tumors affect people of all ages and are the second leading cause of cancer deaths in children. While current treatments are effective and improve survival, there remains a substantial need for more efficacious therapeutic modalities. The ketogenic diet (KD) - a high-fat, low-carbohydrate treatment for medically refractory epilepsy - has been suggested as an alternative strategy to inhibit tumor growth by altering intrinsic metabolism, especially by inducing glycopenia.

Methods: Here, we examined the effects of an experimental KD on a mouse model of glioma, and compared patterns of gene expression in tumors vs. normal brain from animals fed either a KD or a standard diet.

Results: Animals received intracranial injections of bioluminescent GL261-luc cells and tumor growth was followed in vivo. KD treatment significantly reduced the rate of tumor growth and prolonged survival. Further, the KD reduced reactive oxygen species (ROS) production in tumor cells. Gene expression profiling demonstrated that the KD induces an overall reversion to expression patterns seen in non-tumor specimens. Notably, genes involved in modulating ROS levels and oxidative stress were altered, including those encoding cyclooxygenase 2, glutathione peroxidases 3 and 7, and periredoxin 4.

Conclusions: Our data demonstrate that the KD improves survivability in our mouse model of glioma, and suggests that the mechanisms accounting for this protective effect likely involve complex alterations in cellular metabolism beyond simply a reduction in glucose.

Figures

Figure 1

MRI demonstrates the presence of tumors. MRI scans were done on tumor-bearing mice on days 9 and 15. Animals were positioned onto a probe in a head first, prone position. A T2 weighted MRI at 4.7 Tesla was obtained on 16 coronal sections of the animal's frontal lobe at a slice thickness of 0.5 mm.

Figure 2

Animals fed the ketogenic diet showed an increase in blood BHB levels. There was a statistically significant difference by 2-way ANOVA (p = 0.0067) in blood BHB levels in animals fed KD versus SD. There was no difference in the results obtained at the 2 different time points. Five animals were used per condition and the results are shown as the mean ± SEM.

Figure 3

Animals fed a ketogenic diet had a statistically significant increase in survival following tumor implantation. The number in parentheses is the number of animals in each treatment group.

Figure 4

Tumor growth in animals fed a standard diet (black circle) or a ketogenic diet (blue square). Animals were randomized to a treatment arm on day 3 post-implantation. Bioluminescence (photon count) was measured every 3 days. Results are an average of 5 animals for each diet.

Figure 5

Ketones reduced ROS levels. (A) Cells were treated with a cocktail containing either 1 mM (total 2 mM ketones) or 5 mM each (total 10 mM ketones) BHB and AcA for 24 hr prior to analysis. ROS levels were analyzed using DCF as described in the methods. Untreated tumor cells had high levels of ROS, and the application of either 2 mM or 10 mM ketones resulted in a dose dependent statistically significant decrease in the DCF signal demonstrating a reduction of ROS. (* = p ≤ 0.05),(* * = p ≤ 0.001); (B) The presence of increased reactive oxygen species (ROS) in tumor and the surrounding area relative to normal tissue is shown in a mouse fed a standard diet. Dihydroethidium (DHE) was imaged using an excitation wavelength of 500 nm and an emission wavelength of 620 nm. Spectral unmixing was used to differentiate the DHE signal from autofluorescence (shown in purple). The tumor was visualized using bioluminescence (shown in green); (C) Photomicrograph of brain slices from animals fed standard diet (SD) or ketogenic diet (KD). Areas from the tumor core and invading front of the tumor are shown. DCF fluorescence was analyzed as described in the methods section. There is a statistically significant difference in the amount of ROS in tumor vs normal brain when animals fed a standard diet were compared to those fed the ketogenic diet. (* = p ≤ 0.05); N.S. = not significant.

Figure 6

The KD alters overall gene expression to more closely resemble that seen in normal brain. Eight microarrays were analyzed by 2-way ANOVA for interaction effects, using standard Bonferonni multiple testing correction. There were strong interactions between ketogenic diet and normal diet in non-tumor classes, especially in context of standard diet non-tumor. The trend is that many expression profiles in tumor mice on a ketogenic diet seem to trend back to a profile seen with mice living on a standard diet having no tumor.

Figure 7

Expression of genes with significant differential expression in animals fed a KD versus SD. Log2 expression levels of genes that have ≥2-fold expression in at least one condition ratio. 95% confidence intervals are shown.