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, 10 (6), e0129802
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Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model

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

Raphael Johannes Morscher et al. PLoS One.

Abstract

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.

Conflict of interest statement

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

Figures

Fig 1
Fig 1. Ketogenic diet and calorie restriction reduce tumor growth and prolong survival in a NB xenograft model.
After establishing tumors on the right flank of CD-1nu mice, the mice were randomized to diet groups as indicated. Tumor volume was measured twice weekly. A) For SH-SY5Y xenografts at day 19, the tumors of all diet groups showed significant growth inhibition compared to the SD group (CR-SD p = 0.001, KD p<0.001, CR-KD p<0.001). B) At day 33, SK-N-BE(2) tumor growth was significantly inhibited by CR (CR-SD p = 0.040, CR-KD p = 0.004). Inhibition of tumor growth was less pronounced in the KD group (p = 0.918). C) SH-SY5Y and D) SK-N-BE(2) show the results of Kaplan-Meier survival analysis of the corresponding treatment groups. Survival of mice with SH-SY5Y tumors at day 22 on SD was 0% compared to 75% on CR-SD (p<0.001), 50% on KD (p<0.001) and 100% on CR-KD (p<0.001). Survival of mice with SK-N-BE(2) xenografts at day 33 on SD was 36% compared to 83% on CR-SD (p = 0.017), 73% on KD (p = 0.09) and 100% on CR–KD (p<0.001). A, B) Data points for tumor growth curves represent mean values ± SEM of the corresponding diet group (n = 8–11). Statistics: ANOVA (p<0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. C, D) Survival is expressed by the Kaplan–Meier method and differences between groups were determined in a univariate analysis with the log-rank test. Death is coded: tumor volume above 3000 mm3, tumor ulceration or impaired health condition. Diet groups are compared to the corresponding SD. * p≤0.05; ** p≤0.01; *** p≤0.001.
Fig 2
Fig 2. Proliferation indices Ki-67 (A-C) and PHH3 (D-F) suggest reduced proliferation by G0 or early G1 arrest.
CR reduces proliferation in both cell lines whereas KD caused no reduction of proliferative activity in the SK-N-BE(2) xenograft group. A) shows a representative SD Ki-67 staining pattern in comparison to the CR-KD pattern (B) in SK-N-BE(2) tumor samples. For SH-SY5Y xenografts, PHH3 staining is depicted in D) and E) for SD and CR-KD treatment, respectively. Results are given as mean ± SEM. Statistics: ANOVA (p <0.05) followed by Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD. * p≤0.05; ** p≤0.01; *** p≤0.001.
Fig 3
Fig 3. Evaluation of the adaptive response in mtDNA copy number and OXPHOS enzyme activities in NB xenografts to dietary intervention.
Both cell types SH-SY5Y A) and SK-N-BE(2) C) show consistent low copies of the mitochondrial genome (mean <150). Panels B) SH-SY5Y and D) SK-N-BE(2) show mean complex II (succinate dehydrogenase) enzyme activities in xenografts of the different diet groups. Enzyme activities of all individual OXPHOS complexes are additionally given in the S1 and S2 Figs. No significant differences were detected between the different diet groups. For the SK-N-BE(2) xenografts, only representative samples were measured (n≥5). Statistics: ANOVA (p <0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD group. The gray bars represent control values from kidney cortex tissue as reported previously [3].
Fig 4
Fig 4. Western blot analysis of subunits of OXPHOS complexes (CI-CV) from SH-SY5Y (A) and SK-N-BE(2) (B) xenografts.
Three samples from each diet group were loaded as indicated at the top (SD, CR-SD, KD and CR-KD). Representative subunits of CI-CV were probed as given on the left. Both cell types show consistent strong downregulation of CII, with the other complexes being relatively more preserved at the protein level. GAPDH is shown as loading control. As controls (cont1 and cont2), kidney cortex was used as described in [3]. Quantification of loading adjusted staining intensities is given in S3 Fig.
Fig 5
Fig 5. Immunohistochemical (IHC) staining of mitochondrial OXPHOS complexes I-V and VDAC in SH-SY5Y xenograft tumors.
IHC staining of NB sections were scored on a scale from 0–3 as described in the methods section. The CR-KD group showed a significant decrease in complex I staining. All other evaluated parameters were unaffected by dietary changes. Voltage-dependent ion channel (VDAC) protein levels are used as a surrogate marker of mitochondrial mass. Statistics: ANOVA (p <0.05) followed by two-tailed Dunnett’s test correcting for multiple comparisons. Diet groups are compared to the corresponding SD group.

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This work was supported by the Vereinigung zur Förderung der pädiatrischen Forschung und Fortbildung Salzburg, the Children’s Cancer Foundation Salzburg and the Paracelsus Medical University Salzburg, single project grant No.: E-10/12/061-KOF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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