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Link to original content: https://www.ncbi.nlm.nih.gov/pubmed/26083629
The Ketogenic Diet Alters the Hypoxic Response and Affects Expression of Proteins Associated with Angiogenesis, Invasive Potential and Vascular Permeability in a Mouse Glioma Model - PubMed Skip to main page content
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. 2015 Jun 17;10(6):e0130357.
doi: 10.1371/journal.pone.0130357. eCollection 2015.

The Ketogenic Diet Alters the Hypoxic Response and Affects Expression of Proteins Associated with Angiogenesis, Invasive Potential and Vascular Permeability in a Mouse Glioma Model

Eric C Woolf  1 , Kara L Curley  2 , Qingwei Liu  3 , Gregory H Turner  3 , Julie A Charlton  2 , Mark C Preul  4 , Adrienne C Scheck  5
Affiliations

The Ketogenic Diet Alters the Hypoxic Response and Affects Expression of Proteins Associated with Angiogenesis, Invasive Potential and Vascular Permeability in a Mouse Glioma Model

Eric C Woolf et al. PLoS One. .

Abstract

Background: The successful treatment of malignant gliomas remains a challenge despite the current standard of care, which consists of surgery, radiation and temozolomide. Advances in the survival of brain cancer patients require the design of new therapeutic approaches that take advantage of common phenotypes such as the altered metabolism found in cancer cells. It has therefore been postulated that the high-fat, low-carbohydrate, adequate protein ketogenic diet (KD) may be useful in the treatment of brain tumors. We have demonstrated that the KD enhances survival and potentiates standard therapy in a mouse model of malignant glioma, yet the mechanisms are not fully understood.

Methods: To explore the effects of the KD on various aspects of tumor growth and progression, we used the immunocompetent, syngeneic GL261-Luc2 mouse model of malignant glioma.

Results: Tumors from animals maintained on KD showed reduced expression of the hypoxia marker carbonic anhydrase 9, hypoxia inducible factor 1-alpha, and decreased activation of nuclear factor kappa B. Additionally, tumors from animals maintained on KD had reduced tumor microvasculature and decreased expression of vascular endothelial growth factor receptor 2, matrix metalloproteinase-2 and vimentin. Peritumoral edema was significantly reduced in animals fed the KD and protein analyses showed altered expression of zona occludens-1 and aquaporin-4.

Conclusions: The KD directly or indirectly alters the expression of several proteins involved in malignant progression and may be a useful tool for the treatment of gliomas.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Weight, βHB and glucose measurements.
Blood ketone and glucose measurements show (A) higher βHB and (B) lower glucose in KC treated animals (C) weight measurements were taken every 3 days. Graph shows weights normalized to the average starting weight of each group. (N = 5; *p < 0.05; **p < 0.01; ***p < 0.001).
Fig 2
Fig 2. In vivo imaging of hypoxia.
(A) The fluorescent probe HypoxiSense 680 was used to analyze hypoxia in vivo 21 days following tumor implantation. (B) Fluorescent signal was quantitated from tumor bearing mice (N = 5; *p < 0.05). Animals were imaged prior to injection to analyze tissue autofluorescence (“Before injection”; N = 5; ***p < 0.001). Non-tumor bearing mice were injected to analyze non-specific binding (“Normal Brain”; N = 2; *p < 0.05). (C) Tumor bioluminescence imaging showed no significant difference between SD and KC (N = 5).
Fig 3
Fig 3. Western blot analysis of CA IX, HIF-1α, phospho-NF-κB, and total NF-κB.
(A) Western blots showing two representative samples per treatment group. (B) On day 21 post-implantation expression was quantified and represented as a fold change from SD (N = 6; *p < 0.05; **p < 0.01).
Fig 4
Fig 4. Analysis of tumor microvasculature components and gene expression.
(A) CD31 immunostaining of tissue harvested at 21 days post-implantation. Representative images are shown. (B) Quantification of CD31 staining was performed on 2 independent tumors from each group. Data calculated as the average pixel density in 5 random, 200x fields within the same tumor and represented as a fold change from SD (**p < 0.01).
Fig 5
Fig 5. Western blot analysis of CD31, VEGF and VEGFR2.
(A) Western blots showing two representative samples per treatment group. (B) At day 21 post implantation expression was quantified and represented as a fold change from SD (N = 6 for SD and N = 5 for KC; *p < 0.05). (C) Expression of genes involved in angiogenesis was analyzed using RT-PCR. Data represented as the fold change difference in expression from SD tumors (N = 3; *p < 0.05; **p < 0.01).
Fig 6
Fig 6. Western blot analysis of pro- and activated- MMP-2, MMP-9, and vimentin.
(A) Western blots showing two representative samples per treatment group. (B) On day 21 post-implantation expression was quantified and represented as a fold change from SD (N = 6 for SD; N = 5 for KC; *p < 0.05).
Fig 7
Fig 7. Peritumoral edema measurements.
(A) Representative MRI image of tumor bearing mouse (B) 14 days following tumor implantation, edema was measured via MRI. A 2-fold decrease in peritumoral edema was seen in animals fed KC compared to SD (N = 3; *p < 0.05). (C) Tumor bioluminescence imaging showed no significant difference in tumor size between treatments.
Fig 8
Fig 8. Western blot analysis of ZO-1, occludin, aquaporin-1 and Aquaporin-4.
(A) Western blots showing two representative samples per treatment group. (B,C) On day 21 post-implantation expression was quantified and represented as a fold change from SD (N = 6 for SD; N = 5 for KC; *p < 0.05; **p < 0.01).
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This work was supported by an unrestricted grant from Students Supporting Brain Tumor Research (www.ssbtr.org). The authors also received a grant-in-kind from Nutricia Advanced Medical Nutrition through Nutricia North America (http://nutrition.nutricia.com/), who provided KetoCal and the Remi Savioz GLUT1 Foundation (http://www.rsg1foundation.com/) who provided the blood analysis equipment. The funders had no role in data collection and analysis, decision to publish or preparation of the manuscript, or the study design. No additional external funding was received for this study.