A ketogenic diet supplemented with medium-chain triglycerides enhances the anti-tumor and anti-angiogenic efficacy of chemotherapy on neuroblastoma xenografts in a CD1-nu mouse model - PubMed (original) (raw)

A ketogenic diet supplemented with medium-chain triglycerides enhances the anti-tumor and anti-angiogenic efficacy of chemotherapy on neuroblastoma xenografts in a CD1-nu mouse model

Sepideh Aminzadeh-Gohari et al. Oncotarget. 2017.

Abstract

Neuroblastoma (NB) is a pediatric malignancy characterized by a marked reduction in aerobic energy metabolism. Recent preclinical data indicate that targeting this metabolic phenotype by a ketogenic diet (KD), especially in combination with calorie restriction, slows tumor growth and enhances metronomic cyclophosphamide (CP) therapy of NB xenografts. Because calorie restriction would be contraindicated in most cancer patients, the aim of the present study was to optimize the KD such that the tumors are sensitized to CP without the need of calorie restriction. In a NB xenograft model, metronomic CP was combined with KDs of different triglyceride compositions and fed to CD1-nu mice ad libitum. Metronomic CP in combination with a KD containing 8-carbon medium-chain triglycerides exerted a robust anti-tumor effect, suppressing growth and causing a significant reduction of tumor blood-vessel density and intratumoral hemorrhage, accompanied by activation of AMP-activated protein kinase in NB cells. Furthermore, the KDs caused a significant reduction in the serum levels of essential amino acids, but increased those of serine, glutamine and glycine. Our data suggest that targeting energy metabolism by a modified KD may be considered as part of a multimodal treatment regimen to improve the efficacy of classic anti-NB therapy.

Keywords: Warburg effect; ketogenic diet; medium-chain triglycerides; neuroblastoma.

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Conflict of interest statement

CONFLICTS OF INTEREST Tricia Rutherford, Maura O’Donnel, Andrea Stöger-Kleiber are employees of Vitaflo International and therefore have a potential financial conflict of interest. The other authors declare they have no conflicts of interest.

Figures

Figure 1

Figure 1. NB growth was most effectively inhibited by metronomic CP in combination with dietary intervention with LCT-MCT8

Growth suppression of A. SH-SY5Y and B. SK-N-BE(2) xenografts was most pronounced for metronomic CP combined with LCT-MCT8 KD compared to the other KDs and the CRTL diet. Kaplan-Meier survival curves for mice with C. SH-SY5Y and D. SK-N-BE(2) xenografts indicate prolonged survival of the LCT-MCT8 group. Values are given as mean ± SEM (CTRL, LCT and LCT-MCT10, n = 10-12; LCT-MCT8, n = 11-12). One-way ANOVA followed by Dunnett’s Multiple Comparison Test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; KDs in combination with metronomic CP vs CTRL in combination with metronomic CP. The statistical analysis for the survival curves was done with the Log-rank test (Mantel-Cox): SH-SY5Y xenograft, CTRL vs LCT, p = 0.4; CTRL vs LCT/MCT8, p = 0.02; CTRL vs LCT/MCT10, p = 0.02. SK-N-BE(2) xenograft, CTRL vs LCT, p = 0.09; CTRL vs LCT/MCT8, p = 0.002; CTRL vs LCT/MCT10, p = 0.07. When the tumor size reached the termination size some mice had to be sacrificed before the pre-determined end date of the therapeutic intervention. The tumor size data from the last measurement was kept in the calculation for the overall tumor growth rate at later time points.

Figure 2

Figure 2. All KDs significantly lowered blood glucose levels and increased blood ketone body levels

A. The mean blood glucose level significantly decreased and B. the mean BHB level significantly increased in KD-treated mice with NB xenografts over the duration of the experiment. C. The ratio of blood glucose to ketone levels was significantly lower in KDs over the duration of the experiment. Values are given as mean ± SEM (CTRL, LCT and LCT-MCT10, n = 22; LCT-MCT8, n = 23). One-way ANOVA followed by Dunnett’s Multiple Comparison Test; ** p ≤ 0.01; *** p <0.001; KDs in combination with metronomic CP vs CTRL in combination with metronomic CP.

Figure 3

Figure 3. Amino acid levels in plasma and tumor tissue were affected by KDs

Measurement of amino acid levels in A. plasma of mice with NB xenografts and in B. NB tumors (SK-N-BE(2)) revealed significantly lower levels of several essential amino acids and higher levels of the non-essential amino acids serine, glycine and glutamine in the KD groups. Values are given as mean ± SEM (plasma, n = 16; tumor, n = 8). One-way ANOVA followed by Dunnett’s Multiple Comparison Test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; KDs in combination with metronomic CP vs CTRL in combination with metronomic CP. Tryptophan was below the detection limit in tumor tissues.

Figure 4

Figure 4. Levels of urea cycle metabolites were altered by KDs

The level of ornithine (Orn) was significantly reduced in the A. plasma of mice with NB xenografts, and the level of citrulline (Cit) was significantly increased in both the A. plasma of mice with NB xenografts and in B. tumor tissue (SK-N-BE(2)), in the KD groups. Argininosuccinate (Ags) was only detectable in tumors of KD-treated mice. Ammonia (Amm) levels were lower in the KD groups compared to the CTRL group. C. Illustration summarizing the changes in urea metabolites induced by KDs. Values are given as mean ± SEM (plasma, n = 16; tumor, n = 8). One-way ANOVA followed by Dunnett’s Multiple Comparison Test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; KDs in combination with metronomic CP vs CTRL in combination with metronomic CP.

Figure 5

Figure 5. Macroscopic evaluation of intratumoral hemorrhage and microscopic evaluation of tumor vascularization revealed an anti-angiogenic effect of KDs

A. SK-N-BE(2) xenograft tumor homogenates and B. whole tumors showed lower levels of intratumoral hemorrhage in the KD-treated groups compared to the CTRL group. C. Quantification of CD31-positive vessels (counted in five random high-power fields, 100-fold magnification) revealed a significant reduction in vessel density. Representative immunohistochemical staining for the endothelial marker CD-31 in four tumors from the D1, D2 CTRL and D3, D4 LCT-MCT8 groups revealed differences in blood vessel morphology and density. Scale bar = 100 µm. Values are given as mean ± SEM (n = 7-12). One-way ANOVA followed by Dunnett’s Multiple Comparison Test; *** p ≤ 0.001.

Figure 6

Figure 6. Expression of carbonic anhydrase IX (CAIX) was associated with necrosis

A1.-A3. Immunohistochemical staining for CAIX shows the expression of CAIX at the border of necrotic areas in CTRL and LCT-MCT8 groups. B1., B2. Expression of CAIX in non-necrotic tumors in the LCT-MCT8 KD group was lower and focal. C. The mean of the percentage of necrotic areas in SK-N-BE(2) tumors was higher in the CTRL group compared to the LCT-MCT8 group, although this difference was not significant (n = 11). Scale bar = 100 µm.

Figure 7

Figure 7. Western blot analysis of SK-N-BE(2) xenografts showed energy stress and lack of autophagy in LCT-MCT8 group compared to CTRL

Western blot analysis for pAMPK, AMPK and β-Actin in SK-N-BE(2) tumors in the A. LCT-MCT8 and B. LCT groups versus the CTRL group showed activation of AMPK in the KD groups. C. Western blot analysis for pAMPK, AMPK and GAPDH in muscle tissues of mice revealed no difference between the LCT-MCT8 and CTRL groups. D. The ratio of pAMPK to AMPK band intensities indicates higher levels of the activated form of AMPK in the KD groups. The pAMPK and AMPK bands were normalized to the β-Actin levels. E. Western blot analysis for LC3B and β-Actin in SK-N-BE(2) tumors as well as F. the ratio of LC3BI to LCTBII band intensities revealed no difference in autophagy levels between the LCT-MCT8 and CTRL groups. Values are given as mean ± SEM. Unpaired t test; * p ≤ 0.05; ** p ≤ 0.01.

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