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Combination of metronomic cyclophosphamide and dietary intervention inhibits neuroblastoma growth in a CD1-nu mouse model

  • University Hospital Salzburg Paracelsus Medical University Salzburg

Abstract and Figures

Background: MYCN-amplification in high-grade Neuroblastoma (NB) tumors correlates with increased vascularization and therapy resistance. This study combines an anti-angiogenic approach with targeting NB metabolism for treatment. Methods and results: Metronomic cyclophosphamide (MCP) monotherapy significantly inhibited NB growth and prolonged host survival. Growth inhibition was more pronounced in MYCN-amplified xenografts. Immunohistochemical evaluation of this subtype showed significant decrease in blood vessel density and intratumoral hemorrhage accompanied by blood vessel maturation and perivascular fibrosis. Up-regulation of VEGFA was not sufficient to compensate for the effects of the MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were evident. In contrast non MYCN-amplified tumors developed resistance, which was accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or calorie-restriction significantly enhanced the anti-tumor effect. Calorie-restricted ketogenic diet in combination with MCP resulted in tumor regression in all cases. Conclusions: Our data show efficacy of combining an anti-angiogenic cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model. These findings might open a new front in NB treatment.
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Oncotarget17060 Oncotarget, Vol. 7, No. 13
Combination of metronomic cyclophosphamide and dietary
intervention inhibits neuroblastoma growth in a CD1-nu mouse
Raphael Johannes Morscher1,4, Sepideh Aminzadeh-Gohari1, Cornelia Hauser-
Kronberger2, René Günther Feichtinger1, Wolfgang Sperl3, Barbara Koer1
1 Laura Bassi Centre of Expertise-THERAPEP, Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria
2Department of Pathology, Paracelsus Medical University, 5020 Salzburg, Austria
3Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria
4 Division of Medical Genetics, Medical University Innsbruck, 6020 Innsbruck, Austria
Correspondence to: Raphael Johannes Morscher, e-mail:
Keywords: neuroblastoma, ketogenic diet, glucose, metronomic cyclophosphamide, anti-angiogenic
Received: September 15, 2015 Accepted: February 05, 2016 Published: March 05, 2016
Background: MYCN-amplication in high-grade Neuroblastoma (NB) tumors
correlates with increased vascularization and therapy resistance. This study combines
an anti-angiogenic approach with targeting NB metabolism for treatment.
Methods and Results: Metronomic cyclophosphamide (MCP) monotherapy
signicantly inhibited NB growth and prolonged host survival. Growth inhibition was
more pronounced in MYCN-amplied xenografts. Immunohistochemical evaluation
of this subtype showed signicant decrease in blood vessel density and intratumoral
hemorrhage accompanied by blood vessel maturation and perivascular brosis.
Up-regulation of VEGFA was not sufcient to compensate for the effects of the
MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were
evident. In contrast non MYCN-amplied tumors developed resistance, which was
accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or
calorie-restriction signicantly enhanced the anti-tumor effect. Calorie-restricted
ketogenic diet in combination with MCP resulted in tumor regression in all cases.
Conclusions: Our data show efcacy of combining an anti-angiogenic
cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model.
These ndings might open a new front in NB treatment.
Neuroblastoma (NB) represents the most common
extracranial solid childhood cancer. Marked biological
and clinical heterogeneity, pose strong challenges to
optimizing therapeutic interventions for individual
cases. Among others biological risk factors include
MYCN status, tumor histology, cancer cell DNA content
and dened segmental chromosomal aberrations [1–5].
In combination with historical clinical data, tumor biology
allows clinicians to guide therapy by stratifying patients
into internationally accepted risk groups [6, 7]. In low- and
intermediate-risk groups, with overall survival rates above
90%, recent studies have been focusing on reducing
therapeutic toxicity. For patients in the high-risk group,
however, improving treatment efcacy is still central with
overall survival rates close to 50% despite multimodal
therapy protocols [1].
With regard to cellular metabolism NBs share the
characteristic reprogramming to high glucose uptake
recently added to the hallmarks in cancer [8–10]. This
preferential utilization of glucose via aerobic glycolysis
even under sufcient oxygen to shunt pyruvate into
oxidative phosphorylation (OXPHOS) pathway, is
commonly described as Warburg effect [11, 12]. Whereas
defects in single OXPHOS subunits can be a direct
cause of the Warburg effect [13–15], other cancers show
a general pattern of low OXPHOS activity [16–19].
In NB, both primary tumor tissue and xenografts
display generalized low mitochondrial respiratory chain
activity [20, 21]. This metabolic feature might indicate a
dependency on glucose not only for anabolic processes,
but also for energy production. Based on this theory, we
recently reported a preclinical study showing signicant
tumor growth inhibitory effects of dietary intervention in
a NB xenograft model [21]. In that study we observed that
a mild ketogenic diet (KD) and/or calorie restricted (CR)
diet altered blood metabolic parameters (reduced glucose
and increased beta-hydroxybutyrate levels) and induced
signicant growth inhibition of NB xenografts. Detailed
evaluation of mitochondrial OXPHOS parameters showed
no adaptive response in the NB tumors [21].
These growth inhibitory effects are in line with
preclinical data on cancers of the central nervous system
suggesting that CR-KD might constitute an adjuvant
approach in cancer therapy [22–25]. Effects of CR
and/or KD in diverse preclinical cancer models have
recently been reviewed [26, 27]. Anecdotal case reports
of cancer patients treated with CR-KD support a possible
inhibitory effect on tumor growth, but comprehensive
preclinical and clinical evaluation is lacking [28–30]. Pilot
studies of KD in adult patients showed that it might be
tolerated in patients with advanced cancers [31–33].
Because targeting cancer metabolism is
complementary to current treatment strategies for NB,
it could potentially open a new front against this tumor
entity. The current study therefore aims to investigate
the combination of KD and/or CR diet with a low-dose
metronomic cyclophosphamide (MCP) chemotherapy
regimen. MCP has been shown to effectively inhibit
neoangiogenesis in xenograft models of different
tumor entities [34–36] and is under clinical evaluation
[37–40]. We hypothesize that concurrent targeting of
tumor vascularization and cancer cell metabolism might
constitute a cooperative therapeutic approach.
Inhibition of NB tumor growth by MCP in
combination with dietary intervention
In an effort to exploit a potential synergism of
targeting vascular supply and cancer cell metabolism,
we chose to combine dietary intervention with MCP. In
the presented model, MCP induced signicant growth
inhibition (p < 0.001) of NB xenografts of both cell
lines tested from day 9 onward and extended survival
(p < 0.001) compared to the corresponding standard
diet group without MCP (SD group w/o CTx; Figure 1).
Intriguingly on the metronomic dosing protocol,
growth inhibition of SK-N-BE(2) tumors was more
pronounced versus that of tumors of the reportedly more
chemotherapy sensitive SH-SY5Y cell line [41–43].
In addition to the chemotherapy-induced growth reduction,
SH-SY5Y tumors showed a signicant additional
growth inhibition in all three dietary intervention groups
(Figure 1A1). On day 36, tumor volume in the SD group
was signicantly greater compared to that of the calorie
restricted-standard diet (CR-SD) group (p < 0.05), the KD
group (p < 0.01) and the CR-KD group (p < 0.001). In the
SK-N-BE(2) xenografts tumor growth was signicantly
inhibited in the restricted diet groups (Figure 1B1)
CR-SD (p < 0.05) and CR-KD (p < 0.05). Based on the
strong effect of MCP on SK-N-BE(2) tumor growth,
observed absolute effect size of dietary intervention
was limited. One mouse in the CR-SD group was found
dead on day 22 form an undetermined cause. CR-KD
in combination with MCP resulted in tumor regression
all cases of SH-SY5Y and SK-N-BE(2). The fractional
product of Webb [44] on the last day with available control
tumor data showed synergism for KD and CR-KD for
SH-SY5Y and CR-SD and KD for SK-N-BE(2). For the CR-
SD an additive effect and for the CR-KD in SK-N-BE(2)
antagonism was observed (Supplementary Table S3).
These results need to be interpreted with caution due to
the small tumor size in the SK-N-BE(2) and the lack of
dosing curves [44].
Effect of dietary intervention on blood glucose
and ketone body levels
KD and/or CR caused consistent changes in blood
glucose and ketone body levels in mice carrying the two
different xenograft types. Data are given for day 36 or the
last day of therapy (Figure 2A1–2A2 and 2B1–2B2). Blood
glucose levels were signicantly decreased in all four CR
groups when compared to the SD group. In detail, blood
glucose levels in SD group of SH-SY5Y were signicantly
higher when compared to CR-SD (p < 0.05) and CR-KD
(p < 0.01). Ketone body levels were signicantly elevated
in the CR-KD group (p < 0.001) compared to the SD
group, but not in the CR-SD. Glucose in the SK-N-BE(2)
SD group was signicantly higher compared to CR-SD
(p < 0.01) and CR-KD (p < 0.01). Ketone body levels
were signicantly elevated in both the KD (p < 0.05)
and CR-KD (p < 0.001) groups when compared to the
SD group, but not in the CR-SD group. No signicant
changes in blood glucose were detected for neither of the
KD groups. Detailed blood glucose and ketone body data
for all time points is given in Supplementary Table S1. As
published recently [45] the glucose ketone index (GKI), a
ratio of blood glucose [mM] to blood ketone [mM] levels
might help to monitor therapeutic efcacy in patients on
ketogenic diets. Under the given food regimens, mice
showed a GKI > 12 in the SD groups and GKI < 5 in the
CR-KD diet group, on the last day of therapy. These ratios
are consistent with other preclinical studies in mice [45].
Mean GKI is given in Figure 2A3 and 2B3 and shows
a signicant reduction in all dietary intervention groups
(p < 0.001). GKI for all time points are given in
Supplementary Table S2.
Tumor and body weight
As tumor volume calculated by the described
formula could be regarded as an approximation of
tumor size, tumor weight was recorded after sacricing
mice. In line with the tumor volumes, tumor weight was
signicantly decreased in all groups on MCP (p < 0.001).
Dietary intervention signicantly enhanced suppression of
tumor growth, evaluated as tumor weight, in all therapy
groups (p < 0.05) except for the KD group of SK-N-
BE(2) (Figure 2A3 and 2B3). No signicant change in the
tumor volume (mm^3) to weight (mg) ratio was observed
(p > 0.05) (Supplementary Figure S1). Treatment with MCP
did not signicantly change mouse weight on the last day
of therapy in mice on SD or KD (p > 0.05) (Supplementary
Figure S2). The combination of MCP with CR caused a
signicant change in mouse weight for all four therapy
groups when compared to the corresponding SD group
(p < 0.05). For one mouse in the CR-KD group therapy
was discontinued due to progressive weight loss on
day 29. Detailed body weight data for all time points is
given in Supplementary Table S1.
Effect of MCP and dietary intervention on
proliferation indices in NB xenografts
To further elucidate the tumor growth inhibitory
effect of the combination of MCP and/or dietary
intervention, we evaluated proliferative activity by
scoring the markers Ki67 and PHH3. The staining patterns
revealed similar effects of MCP and dietary intervention
on proliferation indices in xenografts of the two cell lines.
Ki67 levels did not differ signicantly when comparing the
non-restricted and CR groups receiving MCP treatment to
the corresponding SD group w/o CTx (p > 0.05; Figure 3A1
and 3B1). The expression of PHH3, a marker for M-phase
of the cell cycle, was signicantly lower in the CR
SH-SY5Y xenografts (p < 0.01, Figure 3A2) as well as
in CR SK-N-BE(2) xenografts (p < 0.001, Figure 3B2).
Proliferation indices for each individual subgroup SD/
Figure 1: Dietary intervention enhances the growth inhibitory effect of MCP on NB xenografts. After establishing tumors,
mice were randomized to therapy and control groups as indicated. For xenografts of both cell lines the MCP regimen signicantly inhibited
tumor growth compared to the SD group w/o CTx (p < 0.001). (A1) SH-SY5Y and (B1) SK-N-BE(2) tumor growth curves. Data points
represent mean values ± SEM of the corresponding therapy group (n = 8–12). (A2) and (B2) show Kaplan–Meier survival curves for mice
with SH-SY5Y and SK-N-BE(2) xenografts respectively. Survival was signicantly prolonged in all therapy groups when compared to
the SD group w/o CTx (p < 0.001). The effect of dietary intervention on tumor growth was evaluated by comparing diet groups to the
corresponding SD on MCP. Signicance levels are given for each dietary intervention group compared to SD on MCP and are stacked from
the group with lowest to highest tumor volume. Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett´s test correcting for multiple
comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Differences in survival 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. Abbrev.: SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
KD and CR-SD/CR-KD are given in Supplementary
Figure S3. Together these results indicate that reduced
cell division might play a role in the growth inhibitory
effect of CR, but does not explain the pronounced effect
of MCP in the more chemotherapy-resistant (SK-N-BE(2))
In vitro 4-hydroperoxycyclophosphamide
(4-HC) sensitivity
Because the in vivo results showed MCP to be more
active against xenografts of the reportedly chemotherapy-
resistant cell line SK-N-BE(2), we compared the in vitro
CP-sensitivity of SH-SY5Y and SK-N-BE(2) to conrm
the data reported in the literature [41–43]. We therefore
titrated 4-HC concentration (active metabolite of CP)
to a dose-range, in which both cell lines showed either
complete cell death (10 nM) or no difference in survival
(0.313 nM) under the conditions tested. At all intermediate
doses SH-SY5Y showed signicantly higher sensitivity
to 4-HC (Supplementary Figure S6). From these data we
hypothesized that, rather than a direct cytotoxic effect,
an indirect effect on xenograft growth may be rendering
SK-N-BE(2) tumors more susceptible to MCP. To our
knowledge this has not been shown in literature before.
Macroscopic observation of intratumoral
Macroscopic inspection and evaluation of photo
documentation showed a tendency to stronger intratumoral
hemorrhage in the SK-N-BE(2) xenografts compared to
SH-SY5Y xenografts (Figure 4A1 and 4B1). We therefore
quantied macroscopic hemorrhage at day 36 or the last
day of therapy (Figure 4A2 and 4B2). In the SK-N-BE(2)
group on SD w/o CTx, 100% (8/8) of tumors showed
pronounced macroscopic hemorrhage. On therapy with
MCP this was reduced to 20% (5/25), which approximates
the level of hemorrhage observed in SH-SY5Y xenografts
(22%, 2/9) without MCP. In the SH-SY5Y xenografts,
MCP reduced macroscopic hemorrhage to a level of 6%
(2/35). Intratumoral hemorrhage did not vary between
dietary subgroups and is given in Supplementary
Figure S4.
Figure 2: Blood glucose reduction and induction of ketosis goes with reduced tumor weight in mice under MCP.
(2A) SH-SY5Y groups and (2B) SK-N-BE(2) groups. (A1) and (B1) CR signicantly reduced blood glucose levels in mice with both
xenograft types. (A2) and (B2) Ketone body levels (beta-hydroxybutyrate) were consistently elevated in the CR-KD groups and the KD
group of SK-N-BE(2). The trend in the KD group of SH-SY5Y did not reach statistical signicance. (A3) and (B3) Mean Glucose Ketone
Index over the treatment period was signicantly reduced in all dietary intervention groups (p < 0.001). (A4) and (B4) Tumor weight was
signicantly reduced in all groups on MCP when compared to the SD group w/o CTx (p < 0.001; data not shown) and dietary intervention
groups as given. The results are consistent with the tumor volumes calculated in Figure 1A and 1B. Data are shown for day 36 or the last
day of therapy. Mean values ± SEM of the corresponding therapy group are given (n = 8–12). Statistics: ANOVA (p < 0.05) followed by
two-tailed Dunnett´s test correcting for multiple comparisons; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbrev.: SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
Effect of MCP on microscopic tumor
vascularization, hemorrhage and vessel
To further substantiate the results from macroscopic
inspection, tumor vascularization and intratumoral
hemorrhage were studied on tumor sections stained
with Hematoxylin/Eosin (Figure 5A1 and 5B1). In line
with the macroscopic observations, vascularization and
hemorrhage were signicantly higher in the SK-N-BE(2)
xenografts compared to SH-SY5Y tumors. Upon MCP
treatment, hemorrhage in both xenograft types was
signicantly reduced (p < 0.01). Along with the signicant
reduction of vascularization in the SK-N-BE(2) xenografts
(p < 0.05), maturation of blood vessels was observed in
xenografts of this cell type (Figure 6B). Angiogenesis
scoring across all subgroups is given in Supplementary
Figure S5. The change in vessel structure seemed even
more fundamental than the reduction of vessel number.
Increased nuclear accumulation of HIF1A was evident in
the SK-N-BE(2) xenografts exposed to MCP. This was
accompanied by central regions of cell death (Figure 5C)
and VEGF up-regulation (Figure 5D). Increased VEGFA
could not compensate for the anti-angiogenic effect of
the MCP therapy. It can be speculated that the marked
increase in diffusion distance (as can be seen in Figure 6B1
and 6B2, right) likely contributes to the increasing tissue
hypoxia and cell death as depicted in Figure 5C.
Endothelial cell marker msCD-31 and Van Gieson
staining were used to further characterize the nature of
the prominent hemorrhage in SK-N-BE(2) xenografts.
msCD-31 staining of SK-N-BE(2) tumors showed a poorly
dened vascular pattern largely lacking both mature
vessel structure and a basal membrane. Blood vessels of
all diameters were strongly invaded by NB tumor cells,
resulting in erythrocyte leakage into the tumor (Figure 6B1
and 6B2, left). Only rare blood vessels in the periphery
of SK-N-BE(2) xenografts showed an intact vessel
structure with prominent basal lamina, as was consistently
observed in the SH-SY5Y xenografts (Figure 6A1 and
6A2 left). Under MCP treatment, however, in addition to
a reduction of overall blood vessel density, a pronounced
maturation of blood vessels was evident in SK-N-
BE(2) tumors (Figure 6B1 and 6B2, right). No tumor
cell invasion into vascular structures was observed in
SK-N-BE(2) xenografts exposed to MCP. This change of
pattern was not observed in SH-SY5Y xenografts, where
both treatment groups showed relatively-well developed
vascular patterns (Figure 6A1 and 6A2). Compared to SD
tumors w/o CTx MCP treated tumors showed a tendency
towards decreased cellularity and reduced nucleus/cytosol
ratio in both SH-SY5Y and SK-N-BE(2) xenografts.
Figure 3: Reduced proliferation contributes to the growth inhibitory effect of CR but not MCP. (3A) SH-SY5Y groups
and (3B) SK-N-BE(2) groups. IHC evaluation of proliferation markers on day 36 or the last day of therapy. (A1) and (B1) The fraction
of Ki67 positive stained cells was not signicantly altered by MCP or diet when compared to SD w/o CTx. (A2) and (B2) In xenografts
of both cell lines, PHH3 positive cells per high power eld were signicantly lower in the CR groups. (A3) and (B3) Exemplary tumor
sections stained for PHH3 from mice on SD w/o CTx (left) and CR-KD (right) are shown. Scale bar = 50 µm. Mean values ± SEM of the
therapy group are given (n ≥ 8). Statistics: ANOVA (p < 0.05) followed by two-tailed Dunnett´s test correcting for multiple comparisons;
**p 0.01; ***p 0.001. Abbrev.: Ad libitum: SD/KD; restricted: CR-SD/CR-KD. SD, standard diet; CR, calorie restriction; KD, ketogenic
diet; w/o CTx, without chemotherapy. Proliferation indices for individual dietary subgroups are given in Supplementary Figure S3.
Diametrically opposed regulation of Bcl-2
protein levels in SH-SY5Y and SK-N-BE(2)
tumors under MCP
For both cell lines used in our study, Bcl-2
expression was described previously [46] and was
conrmed by IHC-staining in our study. Under continuous
MCP treatment, SH-SY5Y xenografts of mice on SD
showed a progressive increase in tumor volumes starting
around treatment day 16. Along with this resistance,
signicantly higher Bcl-2 (p < 0.01; Figure 7A)
expression was evident in xenografts exposed to MCP
treatment compared to SD w/o CTx. This is in line with the
literature for NB tumors, which suggest Bcl-2 upregulation
as a mechanism of chemotherapy resistance under
standard treatment [47, 48]. Consistent with the tumor cell
characteristics [48] (chemotherapy resistant and MYCN
amplied) the SK-N-BE(2) cell line showed increased
basal Bcl-2 protein expression levels in the untreated
group when compared to SH-SY5Y. On MCP treatment,
however, Bcl-2 protein levels were signicantly reduced
in the SK-N-BE(2) xenografts (p < 0.001; Figure 7B).
Whereas Bcl-2 staining was more preserved in
vascularized areas, it was particularly low with increased
distance to blood vessels (Figure 7B2, right). Western
blot analysis (Figure 7C) supports the results from IHC.
Whereas SH-SY5Y tumors exhibited slightly increased
Bcl-2 levels upon prolonged MCP treatment, SK-N-BE(2)
tumors showed decreased levels. Concomitant with the
decrease in Bcl-2 levels, an increase in cleaved caspase 3
was observed in MCP-treated SK-N-BE(2) xenografts.
These results support the higher sensitivity of SK-N-BE(2)
tumors to MCP as observed by tumor volume.
The present study combines two alternative
therapeutic approaches to reduce NB tumor growth in a
preclinical model. In this setting, administering an anti-
angiogenic MCP regimen with dietary intervention was
able to signicantly inhibit growth of NB xenografts of
distinct genetic background and chemotherapy sensitivity.
In line with our previous work that focused
exclusively on the effect of dietary intervention [21], KD
and/or CR reduced glucose availability and increased
blood ketone body levels in mice. Signicant reductions of
Figure 4: Predominant intratumoral hemorrhage in MYCN-amplied tumors is reduced upon MCP treatment.
(4A) SH-SY5Y and (4B) SK-N-BE(2) tumors. Images of xenograft sites, as well as tumors after explantation are shown. Pronounced
macroscopic hemorrhage in the SK-N-BE(2) SD group w/o CTx compared to all other therapy groups of both cell lines was evident.
(A1) and (B1) Specimens from SD w/o CTx compared to SD on MCP are shown. (A2) and (B2) Relative quantication of macroscopically
visible hemorrhagic tumors w/o CTx (SH-SY5Y n = 9, SK-N-BE(2) n = 8) and groups on MCP (SH-SY5Y n = 35, SK-N-BE(2) n = 25).
Intratumoral hemorrhage did not vary between dietary subgroups and is given in Supplementary Figure S4. Abbrev.: SD, standard diet;
w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
tumor growth, in addition to MCP treatment, was evident in
all dietary intervention groups except for the SK-N-BE(2)
xenograft group on KD. Although dietary effects in
the SK-N-BE(2) subgroup should be interpreted with
caution due to the small absolute tumor size, results of
different subgroups align with our previous work [21].
This supports the metabolic dependency of NBs and their
limited capacity to adapt to the change in nutrient supply.
In contrast to other studies on the effect of KD and/
or CR on tumor growth [21, 49–51], we not only observed
growth inhibition, but also regression of tumor volume.
Intriguingly, the combination of MCP and CR-KD
induced a signicant reduction of tumor size and complete
growth arrest of NB xenografts of both cell lines tested.
This highlights the importance of combining dietary
intervention with other therapeutic approaches as it
might sensitize tumors to the cytotoxic effects of these
therapeutics. In line with our data, enhancement of anti-
tumor effect has been described in additional preclinical
studies [52–54].
For NB, increased vascularization has been proposed
to correlate with high-risk tumor stage and poor clinical
outcome [55–57]. Targeting neovascularization has
therefore been put forward as a therapeutic option [58–61].
Furthermore, a direct link between MYCN amplication
and enhanced angiogenesis has been described [55, 62, 63].
In accord we observed increased angiogenesis and a
highly invasive growth pattern in xenografts of the
MYCN-amplied cell line. Upon MCP treatment, this
phenotype changed to a mature vessel pattern with strong
perivascular collagen deposition and without vascular
invasion of tumor cells. To our knowledge this effect has
not been described before. The change in vessel structure
presumably resulted in undersupplied tumors due to
the increased diffusion distance. This interpretation is
supported by the increase in hypoxic areas with central
Figure 5: Microscopic evaluation supports an anti-angiogenic effect of MCP and vessel maturation in NB xenografts.
(5A1) SH-SY5Y and (5B1) SK-N-BE(2). Hematoxylin/Eosin staining of NB sections on day 36 or the last day of therapy was scored for
angiogenesis and hemorrhage on a scale from 0-3. In the SK-N-BE(2) xenografts, blood vessel morphology changed to a more mature
pattern as described in detail in Figure 6B1 and 6B2. (C) IHC staining for HIF1A of a SK-N-BE(2) xenograft section from the SD group
w/o CTx (upper) and SD on MCP (lower). Blood vessel (arrow) rarecation and maturation caused a hypoxic pattern in SK-N-BE(2)
xenografts with increased nuclear HIF1A accumulation and areas of marked cell death in the center of hypoxic areas (asterisk). (D) This
correlated to VEGFA up regulation in SK-N-BE(2) xenografts exposed to MCP, as depicted by immunoblotting. Scale bars = 100 µm
(overview) and 50 µm (detail). Mean values ± SEM of the therapy group are given (n ≥ 8). Statistics: unpaired t test (p < 0.05); *p ≤ 0.05;
**p ≤ 0.01; ***p 0.001. Angiogenesis scoring across all subgroups is given in Supplementary Figure S5. Densitometry for VEGFA levels
is given in Supplementary Figure S8A. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy; MCP, metronomic cyclophosphamide.
cell death as seen on HIF1A staining. Direct evidence for
nutrient deprivation, however, is pending.
Whereas the SH-SY5Y xenografts in the SD group
developed resistance to the MCP regimen that was
accompanied by up-regulation of Bcl-2 levels, in the
reportedly more chemotherapy-resistant SK-N-BE(2)
xenografts [41–43], sensitivity was retained. The increased
in vitro chemotherapy resistance of the SK-N-BE(2)
cells supports an anti-tumor mechanism of MCP that is
not linked to a direct cytotoxic effect. Previous reports
showed that in vitro hypoxia can trigger an autocrine loop
via increased VEGF expression to induce Bcl-2 mediated
chemotherapy resistance in SK-N-BE(2) cells [64, 65].
Although our model supports ndings of increased
VEGFA expression in response to hypoxia, this loop was
not sufcient to induce Bcl-2-mediated tumor cell survival
in the in vivo setting.
This nding might highlight the importance of a
concomitant decrease in nutrient supply that accompanies
the state of low vascularization in tumors which is often
not prioritized in in vitro studies [64, 65]. Supporting
published work on an anti-angiogenic effect of KD [66],
we observed the strongest reduction of angiogenesis in the
combination of KD with MCP. Whereas both publications
report a rarecation of blood vessels and hemorrhage,
the brous depositions and vessel maturation are only
described in our model.
Figure 6: Evaluation of blood vessels by IHC for msCD-31 endothelial cell marker (A1, B1) and Van Gieson staining
of collagen bers (A2, B2). Images show exemplary sections of (6A) SH-SY5Y and (6B) SK-N-BE(2) xenografts at day 36 or the last
day of therapy. The left upper (overview) and lower (detail) images are taken from tumors w/o CTx, the right upper and lower images are
from tumors exposed to MCP. (A1) and (B1) show IHC staining for the endothelial marker msCD-31. Under both conditions SH-SY5Y
xenografts show well-formed blood vessels, as can be appreciated best in the detail section (A1, lower). In comparison SK-N-BE(2)
xenografts show marked difference in blood vessel morphology between the w/o CTx group and the MCP group (B1). (A2) and (B2)
Van Gieson staining identied the strong perivascular connective tissue in the SK-N-BE(2) group on MCP as collagen deposition (red).
Blood vessels are marked by arrows, Scale bars = 100 µm (overview) and 50 µm (detail). Abbrev.: SD, standard diet; w/o CTx, without
chemotherapy; MCP, metronomic cyclophosphamide.
Although we are aware of the limitations of
an ectopic xenograft model and potential effects of
additional molecular differences between the cell lines,
the pronounced effect on tumor growth inhibition and
blood vessel maturation could help to further clarify
mechanisms in this molecular subgroup of NBs [62].
It can be hypothesized, that along with the increased
vascularization, also a stronger dependency on vascular
supply is present for promoting tumor growth. Together
with targeting cell metabolism this might constitute an
Achilles heel worth aiming for to improve treatment for
therapy-refractory NB.
Figure 7: SH-SY5Y (A) and SK-N-BE(2) (B) cell lines show divergent regulation of Bcl-2 levels under MCP. (A1) and
(B1) Bcl-2 levels on IHC staining were scored on day 36 or the last day of therapy. In the untreated state, SH-SY5Y xenografts showed
signicantly lower Bcl-2 levels when compared to SK-N-BE(2) xenografts (p < 0.001). On MCP treatment, the two cell lines showed an
opposing response. (A2) and (B2) Exemplary Bcl-2 IHC-stained sections w/o CTx (left) and on MCP (right). (C) On western blot analysis,
along with the reduction in Bcl-2 levels, increased cleaved caspase 3 protein was detected in the SK-N-BE(2) xenografts exposed to MCP.
Densitometry for western blots is given in Supplementary Figure S8B.Scale bar = 100 µm; Mean values ± SEM of the therapy group are
given (n ≥ 8). Statistics: Stundent´s t test (p < 0.05); **p ≤ 0.01, ***p 0.001. Abbrev.: SD, standard diet; w/o CTx, without chemotherapy;
MCP, metronomic cyclophosphamide.
Cell lines
Neuroblastoma xenografts were established with
the cell lines (ATCC, Manassas, VA, USA) SH-SY5Y
(CRL-2266) and SK-N-BE(2) (CRL-2271). SH-SY5Y
is a TP53 wild type, non-MYCN-amplied cell line with
no chromosome 1p loss of heterozygosity and is sensitive
to chemotherapy. The SK-N-BE(2) cell line is highly
resistant to a wide range of chemotherapeutic agents and
is characterized by MYCN-amplication, TP53 mutation
(p.C135F) and chromosome 1p loss of heterozygosity [41].
Cells were cultured in standard conditions as described
earlier [21]. MYCN expression on protein level was
conrmed on western blot and is given in Supplementary
Figure S7.
Animal models and sample preparation
All in vivo experiments were performed in
accordance with protocols approved for this study by the
Salzburg Animal Care and Use Committee (Nr.: 20901-
TVG/44/7-2011). This protocol specically addressed
weight loss observed in response to dietary intervention.
Animals were maintained under specic pathogen-free
conditions and care conformed to the Austrian Act on
Animal Experimentation. Xenograft growth was induced
on the right anks of 5-to 6-week-old female CD-1
nude mice (Charles River, Wilmington, MA, USA) by
subcutaneous injection of a 200 µl suspension of NB
cells (2.7 × 107) in serum-free medium and matrigel
(BD Bioscience, Franklin Lakes, NJ, USA). After
reaching a tumor size of 350 mm3, an oral metronomic
cyclophosphamide treatment was started and mice were
randomized to four dietary therapy groups (SD, CR-
SD, KD and CR-KD; n = 10–12). One control group on
standard diet without chemotherapy was included for both
cell lines (SD w/o CTx; n = 8–9).
Two times per week, tumor volumes were measured
using a caliper and calculated according to the formula
width x height x length / 2. Body weight was recorded and
blood glucose and ketone body (beta-hydroxybutyrate)
levels were monitored using enzyme based methods twice
weekly (Precision Xceed, Abbott Laboratories, North
Chicago, IL). In the ad libitum-fed groups, measurements
were performed after a two-hour fasting period or before
feeding in the calorie restricted groups. Animals in CR
groups were single-housed to control food intake. Ad
libitum-fed mice were group housed. Mice were sacriced
at day 36 or when termination criteria were met (health
status, tumor ulceration or volume of 3000 mm
). Images
of the xenografts were recorded at the start of therapy,
and before and after removal of the tumor. Cancer tissue
was snap frozen in liquid nitrogen. One 0.5 cm tumor
slice each was formalin-xed and parafn embedded for
histological analysis.
Food composition and energy content
Mice were fed according to four different regimens:
standard diet ad libitum (SD), calorie restricted standard
diet (CR-SD), long-chain fatty acid-based ketogenic diet
ad libitum (KD) and calorie-restricted ketogenic diet
(CR-KD). Detailed food composition and feeding
protocols to evaluate the dietary intervention on NB-tumor
growth were published previously [21] and are given in the
Supplementary Data (Table S4). In brief, the metabolizable
energy contents were: SD (kcal: fat 9%, protein 33% and
carbohydrates 58%) and mild KD (kcal: fat 78%, protein
14% and carbohydrates 8%) (No. V1535-000 and No.
S9139-E02D; Sniff Spezialdiäten GmbH, Soest, D). Diets
were fortied with vitamins and mineral supplements. For
the CR groups, food intake was restricted to 2/3 of the
respective ad libitum intake, which aligns with clinical
protocols for therapy-resistant epilepsy [67–69].
Metronomic cyclophosphamide (MCP) schedule
CP (Sigma, St Louis, MO, USA) was administered
orally through the drinking water as described previously
[70]. In order to achieve the described oral dose of 40 mg/kg
CP per day, nal CP concentration in drinking water was
0.266 mg/ml [70]. Analogous MCP regimens were applied
to target neoangiogenesis in xenograft models of other
tumor entities [34–36] and have low toxicities [70, 71].
Average water/CP intake per mouse was equal between
different diet groups.
In vitro CP sensitivity
In vitro CP sensitivity of both cell lines was
evaluated by utilizing 4-hydroperoxycyclophosphamide
(4-HC, Niomech, Bielefeld, D), an active metabolite of CP.
Cells were seeded in 96 well plates (25 000/well). After
resting for 48 hours, standard medium was replaced by 100
µl medium containing 4-HC (0.157 – 10 nM). 4-HC was
freshly dissolved in DMSO (Sigma), and control medium
contained the maximum DMSO concentration (0.01%).
After three hours of drug exposure, the medium was
replaced by 100 µl standard medium for 24 hours to allow
the cytotoxicity to fully develop. 3-(4,5-dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
was performed following the manufacturer’s instructions
(Life Technologies, Carlsbad, CA, USA). Absorbance
was read at 570 nm with an EnSpire Multimode Plate
Reader (PerkinElmer, Waltham, MA, USA). Results were
normalized to background and are given as metabolic
activity relative to control (100%). The average of two
independent experiments with 6 replicates each is shown.
(Immuno-)histochemical staining and analysis
Histological staining was performed using
5-µm deparafnized tumor sections of NB xenografts.
Hematoxylin/eosin and Van Gieson staining as well as
immunohistochemistry (IHC) stainings (Bcl-2 (B-cell
lymphoma 2), PHH3 (phospho-histone H3), Ki67) were
carried out within the routine diagnostic setting at the
local pathology department following standard protocols.
Antibodies were used at the following dilutions: 1:100
for anti-Bcl-2 (Dako, Glostrup, Denmark), 1:200 for
anti-PHH3 (Cellmarque, Rocklin, CA, USA) and 1:500 for
anti-Ki67 (Dako). Angiogenesis, intratumoral hemorrhage
and Bcl-2 staining were scored on a scale of 0 (none) to 3
(strong). Proliferation indices were scored by evaluating
the proportion of positively stained nuclei scoring at
least 500 cells per slide (Ki67) or counting the number
of positively stained tumor nuclei per high-power eld
(PHH3/HPF). For adequately sized tumors, at least ve
representative regions were scored; magnication was
400-fold. When the tumor size was too small, stained
nuclei/total tumor cells were counted and converted to a
HPF equivalent. Anti-msCD31 (Abcam, Cambridge, UK)
and anti-HIF1A (hypoxia-inducible factor 1-alpha, Novus,
Littleton, USA) stainings were carried out at a dilution
of 1:50 and 1:100, respectively, following protocols for
IHC-staining described earlier [19, 72]. For detection
EnVision kit reagents (Dako) were used following the
manufacturer’s instructions. Analysis of histological
staining was performed by two blinded investigators, and
the mean of both scores was calculated.
Western blot analysis
Whole-cell lysates for western blot analysis were
prepared using standard radioimmunoprecipitation
assay buffer. In brief, 30 µg protein were separated on
10% acrylamide/bis-acrylamide gels before transfer
to polyvinylidene diuoride membranes (Bio-Rad,
Hercules, CA, USA) using CAPS buffer (10 mM
3-[cyclohexylamino]-1-propane sulfonic acid, pH 11; 10%
methanol). The following primary antibodies were diluted
in 5% w/v BSA (Sigma) in TBS-T: 1:1000 anti-Bcl-2
(Dako), 1:1000 anti-cleaved caspase 3 (Cell Signaling
Technologies, Danvers, MA, USA), 1:30 000 anti-tubulin
(Promega, Madison, WI, USA), 1:1000 anti-VEGFA
(vascular endothelial growth factor, Abcam) and 1:1000
anti-NMYC (Abcam). Horseradish peroxidase-labeled
secondary antibodies were used (Dako) and detection
was carried out with Lumi-Light POD-substrate (Roche,
Basel, Switzerland). Quantication of band intensities was
performed using Image Lab Software 5.2.1 (Bio-Rad) and
adapted to the corresponding loading control.
Statistical analysis was performed by Student´s t test
(two groups; p < 0.05) and one-way ANOVA (more than
two groups; p < 0.05). To adapt for multiple comparisons
two-tailed Dunnett´s posttest was used. In these cases
multiplicity adjusted p-value is given. If not mentioned
otherwise, results are given as mean ± SEM. The analyses
were performed using Prism 6 (GraphPad Software, La Jolla,
CA, USA) and SPSS 21 (IBM, Armonk, NY, USA). The
fractional product of Webb was used to determine whether
the observed effects show synergistic or additive nature.
The method uses the formula: i1,2 = i1*i2; where i1,2 is the
inhibitory fraction of the combination of two interventions
i1 (cyclophosphamide) and i2 (diet). When the predicted
response exceeds the measured response, synergism is
claimed (Ratio calculated/hypothetical < 1) [44].
NB, neuroblastoma; SD, standard diet; CR, calorie
restriction; KD, ketogenic diet; w/o CTx, without
chemotherapy; MCP, metronomic cyclophosphamide.
The authors thank PD Dipl Ing. Dr. J. A. Mayr
for fruitful discussions and M. Prinz und B. Lechner for
excellent technical assistance performing IHC-stainings.
This work was supported by the Vereinigung zur
Förderung der pädiatrischen Forschung und Fortbildung
Salzburg, the Children’s Cancer Foundation Salzburg, the
Paracelsus Medical University Salzburg, single project
grant No.: E-10/12/061-KOF and the Austrian Research
Promotion Agency (822782/THERAPEP). The funders
had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
The authors declare that no competing interests
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... Although metronomic topotecan and vinblastine are the chemotherapeutic drugs that have been most frequently used in preclinical models of neuroblastoma, other metronomic therapeutic approaches have been tested with metronomic cyclophosphamide (mCTX) at the dose of 40 mg/kg/day p.o. in SH-SY5Y (chemotherapy-sensitive, non-MYCN-amplified) and SK-N-BE2 (chemotherapy-resistant, MYCN-amplified) tumor xenografts. Morscher and colleagues [32] observed a greater inhibition of tumor expansion in MYCN-amplified mCTX-treated xenografts, as well as a considerable diminution of blood vessel density and intratumoral bleeding. Moreover, they detected a decreased Bcl-2 expression and elevated caspase-3 cleavage. ...
... Moreover, they detected a decreased Bcl-2 expression and elevated caspase-3 cleavage. In contrast, non-MYCN-amplified tumors showed upregulation of Bcl-2 and developed resistance [32]. Intriguingly, combining mCTX with a calorie-restricted ketogenic diet [33] significantly enhanced the antitumor effects of this therapeutic approach, resulting in tumor regression and the complete growth arrest of both NB xenografts [32]. ...
... In contrast, non-MYCN-amplified tumors showed upregulation of Bcl-2 and developed resistance [32]. Intriguingly, combining mCTX with a calorie-restricted ketogenic diet [33] significantly enhanced the antitumor effects of this therapeutic approach, resulting in tumor regression and the complete growth arrest of both NB xenografts [32]. However, calorie restriction would not be recommended in most young oncologic patients. ...
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Metronomic chemotherapy (MC) is the frequent, regular administration of drug doses designed to maintain a low, but active, range of concentrations of chemotherapeutic drugs, during prolonged periods of time without inducing excessive toxicities. To date, more than 400,000 children and adolescents under the age of 20 are diagnosed with cancer, per year, with 80% survival in most high-income countries, but less than 30% in low- and middle-income ones. In this review, we summarized the principal preclinical and clinical studies involving the use of MC in the most common pediatric tumors, with an overview of efficacy, toxicity, pharmacokinetic profile, and biomarkers. The best advantages of MC are low toxicity, oral administration and, thus, the feasibility of a more comfortable, home-based treatment, therefore improving the quality of life of the children themselves and of their parents and caregivers. Moreover, MC could represent a valid method to reduce the economic burden of anticancer therapy in the pediatric setting.
... 64,81,82 KD increases the chemotherapy effect but this increase is much more significant when it is combined with calorie restriction. 83 In this regard, metronomic cyclophosphamide (MCP) combined with CR-KD significantly decreases tumor size and inhibits the growth of NB xenografts. Studies also show that dietary intervention increases the sensitivity of tumor cells to the cytotoxic effects of therapeutic approaches. ...
... Studies also show that dietary intervention increases the sensitivity of tumor cells to the cytotoxic effects of therapeutic approaches. 55,[83][84][85] Nevertheless, although calorierestricted KD can be helpful, more research is required. ...
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The ketogenic diet (KD) was initially used in 1920 for drug-resistant epileptic patients. From this point onward, ketogenic diets became a pivotal part of nutritional therapy research. To date, KD has shown therapeutic potential in many pathologies such as Alzheimer's disease, Parkinson's disease, autism, brain cancers, and multiple sclerosis. Although KD is now an adjuvant therapy for certain diseases, its effectiveness as an antitumor nutritional therapy is still an ongoing debate, especially in Neuroblastoma. Neuroblastoma is the most common extra-cranial solid tumor in children and is metastatic at initial presentation in more than half of the cases. Although Neuroblastoma can be managed by surgery, chemotherapy, immunotherapy, and radiotherapy, its 5-year survival rate in children remains below 40%. Earlier studies have proposed the ketogenic diet as a possible adjuvant therapy for patients undergoing treatment for Neuroblastoma. In this study, we seek to review the possible roles of KD in the treatment of Neuroblastoma.
... The CP dose was selected to induce a mild but not significant effect on tumor growth, in order to enable the evaluation of any additive or synergistic effects of the different therapeutic interventions. The SKNBE(2) xenografts were treated with 13 mg/kg/day according to our previously published protocol [30], and KELLY xenografts were treated with 20 mg/kg/day ( Figure S1). The selected dose of MET (100 mg/kg) was based on dose response experiments initially performed in the SKNBE(2) and KELLY xenografts ( Figure S2). ...
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Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.
... In contrast, my meta-analysis has included survival outcomes from a total of 65 experiments which uniformly supplied a KD in unrestricted amounts. Application of restricted KDs to mice has mostly been studied for central nervous system tumors where was found to be more effective against tumor growth than feeding an unrestricted KD [57,63,68]. The group of Thomas Seyfried and Purna Mukherjee has shown that calorie-restricted KDs are highly effective in slowing down the growth of high-grade gliomas in mice, especially when combined with additional metabolic treatments such as 2-deoxy-DD-glucose [83] or 6diazo-5-oxo-L-norleucine (DON) which antagonizes glutamine, the second fermentable fuel used by cancer cells besides glucose [84]. ...
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Background Ketogenic diets (KDs) are high-fat diets with putatively anti-tumor effects. The aim of this study was to synthesize the evidence for anti-tumor effects of KDs in mice, with a focus on their possible synergism with chemotherapy (CT), radiotherapy (RT) or targeted therapies (TT). Materials and methods Relevant studies were retrieved from a literature search. A total of 43 articles reporting on 65 mouse experiments fulfilled the inclusion criteria, and 1755 individual mouse survival times were collated from the study authors or the publications. The restricted mean survival time ratio (RMSTR) between the KD and control group served as the effect size. Bayesian evidence synthesis models were used to estimate pooled effect sizes and to assess the impact of putative confounders and synergism between KD and other therapies. Results Overall, there was a significant survival-prolonging effect of KD monotherapy (RMSTR=1.161±0.040), which was confirmed in meta-regression accounting for syngeneic versus xenogeneic models, early versus late KD start and subcutaneous versus other organ growth. Combining the KD with RT or TT, but not CT, was associated with a further 30% (RT) or 21% (TT) prolongation of survival. An analysis accounting for 15 individual tumor entities showed that KDs exerted significant survival-prolonging effects in pancreatic cancer (all treatment combinations), gliomas (KD+RT and KD+TT), head and neck cancer (KD+RT) and stomach cancer (KD+TT). Conclusions This analytical study confirmed the overall anti-tumor effects of KDs in a large number of mouse experiments and provides evidence for synergistic effects with RT and TT.
... Moreover, STAT3 could regulate the activity and stability of HIF-1α. Morscher et al. observed that KD inhibited angiogenesis and tumor growth in vivo by suppressing VEGFA [78]. In the present study, we found that DOKD may exert its role in inhibiting CT26 + tumor angiogenesis by inhibiting the STAT3/HIF-1α/VEGFA pathway. ...
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Colon cancer is one of the typical malignant tumors, and its prevalence has increased yearly. The ketogenic diet (KD) is a low-carbohydrate and high-fat dietary regimen that inhibits tumor growth. Donkey oil (DO) is a product with a high nutrient content and a high bioavailability of unsaturated fatty acids. Current research investigated the impact of the DO-based KD (DOKD) on CT26 colon cancer in vivo. Our findings revealed that DOKD administration significantly lowered CT26+ tumor cell growth in mice, and the blood β-hydroxybutyrate levels in the DOKD group was significantly higher than those in the natural diet group. Western blot results showed that DOKD significantly down-regulated Src, hypoxia inducible factor-1α (HIF-1α), extracellular signal-related kinases 1 and 2 (Erk1/2), snail, neural cadherin (N-cadherin), vimentin, matrix metallopeptidase 9 (MMP9), signal transducer and activator of transcription 3 (STAT3), and vascular endothelial growth factor A (VEGFA), and it significantly up-regulated the expressions of Sirt3, S100a9, interleukin (IL)-17, nuclear factor-kappaB (NF-κB) p65, Toll-like receptor 4 (TLR4), MyD88, and tumor necrosis factor-α. Meanwhile, in vitro validation results showed that LW6 (a HIF-1α inhibitor) significantly down-regulated the expressions of HIF-1α, N-cadherin, vimentin, MMP9, and VEGFA, which supported those of the in vivo findings. Furthermore, we found that DOKD inhibited CT26+ tumor cell growth by regulating inflammation, metastasis, and angiogenesis by activating the IL-17/TLR4/NF-κB p65 pathway and inhibiting the activation of the Src/HIF-1α/Erk1/2/Snail/N-cadherin/Vimentin/MMP9 and Erk1/2/HIF-1α/STAT3/VEGFA pathways. Our findings suggest that DOKD may suppress colon cancer progression and help prevent colon cancer cachexia.
... A boost in animal research can be recognized with an apparent diversity in types of cancer being investigated. These encompass gastric [24,93], breast [21,[94][95][96], brain [11,17,87,[97][98][99][100][101][102][103], lung [19,104,105], liver [106,107], colorectal [25,26,28,108], prostate [90,92,[109][110][111], renal [112], thyroid [113], pancreatic [8,114], and melanoma [115] cancers. Based on our review of the literature, brain cancer comes in the lead with malignant glioma being more repeatedly studied. ...
Background: Despite recent advances that have been made in cancer treatment, we still lack complementary approaches to significantly improve the effectiveness of current anti-cancer therapies. The high-fat, low-carbohydrate ketogenic diet can cause metabolic changes in the tumor microenvironment, thus targeting cancer metabolism, making it an attractive candidate for adjuvant cancer therapy. This review is an attempt to explore the origin and trace the historical use of the ketogenic diet as a metabolic therapy in medical oncology. Methods: A detailed literature review was conducted through specialized databases. Only those articles pertinent to the specific needs of the review were included. Relevant references in those articles were also followed. Results: There is a sound biological rationale for the anti-tumor force of the ketogenic diet primarily manifested through its anti-Warburg effect. It is no less true that Otto Warburg's experiments have based the ketogenic diet in cancer research on solid footing. However, precisely tracing the initial, original anti-tumor use of the keto-genic diet in medical oncology literature has indeed proved to be a hard needle to thread. The currently available data are considered promising in the pre-clinical rodent models. Yet, the number of rigorous human trials is small and suffers from methodological limitations. Conclusions: Further research, both pre-clinical and clinical, is needed to accurately define the anti-tumor effect of the ketogenic diet and to overcome the current methodological limitations that hinder us from deriving a sound judgment about the use of the ketogenic diet in clinical practice.
... Furthermore reducing blood glucose is also associated with reduced levels of insulin and insulin-like growth factor, which play an important role in cancer cell proliferation [7]. Numerous preclinical studies have provided evidence for an anti-tomour effect of KD [5,6,[8][9][10][11][12]. The clinical potential of KD in the treatment of cancer has only recently been explored. ...
Background: Neuroblastoma is a paediatric malignancy of incredibly complex aetiology. Oncogenic protein kinase signalling in neuroblastoma has conventionally focussed on transduction through the well-characterised PI3K/Akt and MAPK pathways, in which the latter has been implicated in treatment resistance. The discovery of the receptor tyrosine kinase ALK as a target of genetic alterations in cases of familial and sporadic neuroblastoma, was a breakthrough in the understanding of the complex genetic heterogeneity of neuroblastoma. However, despite progress in the development of small-molecule inhibitors of ALK, treatment resistance frequently arises and appears to be a feature of the disease. Moreover, since the identification of ALK, several additional protein kinases, including the PIM and Aurora kinases, have emerged not only as drivers of the disease phenotype, but also as promising druggable targets. This is particularly the case for Aurora-A, given its intimate engagement with MYCN, a driver oncogene of aggressive neuroblastoma previously considered 'undruggable.' Scope of review: Aided by significant advances in structural biology and a broader understanding of the mechanisms of protein kinase function and regulation, we comprehensively outline the role of protein kinase signalling, emphasising ALK, PIM and Aurora in neuroblastoma, their respective metabolic outputs, and broader implications for targeted therapies. Major conclusions: Despite massively divergent regulatory mechanisms, ALK, PIM and Aurora kinases all obtain significant roles in cellular glycolytic and mitochondrial metabolism and neuroblastoma progression, and in several instances are implicated in treatment resistance. While metabolism of neuroblastoma tend to display hallmarks of the glycolytic "Warburg effect," aggressive, in particular MYCN-amplified, tumours retain functional mitochondrial metabolism, allowing for survival and proliferation under nutrient stress. Future strategies employing specific kinase inhibitors as part of the treatment regimen should consider combinatorial attempts at interfering with tumour metabolism, either through metabolic pathway inhibitors, or by dietary means, with a view to abolish metabolic flexibility that endows cancerous cells with a survival advantage.
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The ketogenic diet (KD) is a high-fat, adequate-protein and very low-carb diet regimen that simulates/mimics the metabolism of the fasting state to stimulate the production of ketone bodies. KD has long stood out as a highly successful dietary approach for the treatment of epilepsy. According to the latest data, the therapeutic effect of KD seems promising for many diseases such as cancer, diabetes, obesity as well as epilepsy. Given the widespread practice of this diet, even among individuals who do not need to lose weight, there is some concern about the potential long-term consequences of widespread adoption of this diet by large sections of the population. In this review, prominent concepts related to KD and the KD are expressed, and experimental and/or clinical data on the efficacy and safety of KD in different diseases are summarized. The possible mechanisms of the understood effects of KD at the cellular and molecular level were discussed, emphasizing that there may be multiple mechanisms that need further clarification. Scientific data on the therapeutic effects and mechanisms of action of KD are insufficient, and more studies are required in order to expand the clinical application of KD.
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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.
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Metabolic therapy using ketogenic diets (KD) is emerging as an alternative or complementary approach to the current standard of care for brain cancer management. This therapeutic strategy targets the aerobic fermentation of glucose (Warburg effect), which is the common metabolic malady of most cancers including brain tumors. The KD targets tumor energy metabolism by lowering blood glucose and elevating blood ketones (β-hydroxybutyrate). Brain tumor cells, unlike normal brain cells, cannot use ketone bodies effectively for energy when glucose becomes limiting. Although plasma levels of glucose and ketone bodies have been used separately to predict the therapeutic success of metabolic therapy, daily glucose levels can fluctuate widely in brain cancer patients. This can create difficulty in linking changes in blood glucose and ketones to efficacy of metabolic therapy. A program was developed (Glucose Ketone Index Calculator, GKIC) that tracks the ratio of blood glucose to ketones as a single value. We have termed this ratio the Glucose Ketone Index (GKI). The GKIC was used to compute the GKI for data published on blood glucose and ketone levels in humans and mice with brain tumors. The results showed a clear relationship between the GKI and therapeutic efficacy using ketogenic diets and calorie restriction. The GKIC is a simple tool that can help monitor the efficacy of metabolic therapy in preclinical animal models and in clinical trials for malignant brain cancer and possibly other cancers that express aerobic fermentation.
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Based on the hypothesis that cancer cells may not be able to metabolize ketones as efficiently as normal brain cells, the ketogenic diet (KD) has been proposed as a complementary or alternative therapy for treatment of malignant gliomas. We report here our experience in treating two glioma patients with an IRB-approved energy-restricted ketogenic diet (ERKD) protocol as monotherapy and review the literature on KD therapy for human glioma patients. An ERKD protocol was used in this pilot clinical study. In addition to the two patients who enrolled in this study, we also reviewed findings from 30 other patients, including 5 patients from case reports, 19 patients from a clinical trial reported by Rieger and 6 patients described by Champ. A total of 32 glioma patients have been treated using several different KD protocols as adjunctive/complementary therapy. The two patients who enrolled in our ERKD pilot study were monitored with twice daily measurements of blood glucose and ketones and daily weights. However, both patients showed tumor progression while on the ERKD therapy. Immunohistochemistry reactions showed that their tumors had tissue expression of at least one of the two critical mitochondrial ketolytic enzymes (succinyl CoA: 3-oxoacid CoA transferase, beta-3-hydroxybutyrate dehydrogenase 1). The other 30 glioma patients in the literature were treated with several different KD protocols with varying responses. Prolonged remissions ranging from more than 5 years to 4 months were reported in the case reports. Only one of these patients was treated using KD as monotherapy. The best responses reported in the more recent patient series were stable disease for approximately 6 weeks. No major side effects due to KD have been reported in any of these patients. We conclude that 1. KD is safe and without major side effects; 2. ketosis can be induced using customary foods; 3. treatment with KD may be effective in controlling the progression of some gliomas; and 4. further studies are needed to determine factors that influence the effectiveness of KD, whether as a monotherapy, or as adjunctive or supplemental therapy in treating glioma patients. NCT01535911.
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To support high proliferation, the majority of cancer cells undergo fundamental metabolic changes such as increasing their glucose uptake and shifting to glycolysis for ATP production at the expense of far more efficient mitochondrial energy production by oxidative phosphorylation (OXPHOS), which at first glance is a paradox. This phenomenon is known as the Warburg effect. However, enhanced glycolysis is necessary to provide building blocks for anabolic growth. Apart from the generation of ATP, intermediates of glycolysis serve as precursors for a variety of biosynthetic pathways essential for cell proliferation. In the last 10-15 years the field of tumor metabolism has experienced an enormous boom in interest. It is now well established that tumor suppressor genes and oncogenes often play a central role in the regulation of cellular metabolism. Therefore, they significantly contribute to the manifestation of the Warburg effect. While much attention has focused on adult solid tumors, so far there has been comparatively little effort directed at elucidation of the mechanism responsible for the Warburg effect in childhood cancers. In this review we focus on metabolic pathways in neuroblastoma (NB) and Wilms tumor (WT), the two most frequent solid tumors in children. Both tumor types show alterations of the OXPHOS system and glycolytic features. Chromosomal alterations and activation of oncogenes like MYC or inactivation of tumor suppressor genes like TP53 can in part explain the changes of energy metabolism in these cancers. The strict dependence of cancer cells on glucose metabolism is a fairly common feature among otherwise biologically diverse types of cancer. Therefore, inhibition of glycolysis or starvation of cancer cells through glucose deprivation via a high-fat low-carbohydrate diet may be a promising avenue for future adjuvant therapeutic strategies.
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The role of dietary restriction regimens such as caloric restriction, ketogenic diet and intermittent fasting in development of cancers has been detected via abundant preclinical experiments. However, the conclusions are controversial. We aim to review the relevant animal studies systematically and provide assistance for further clinical studies. Literatures on associations between dietary restriction and cancer published in PubMed in recent twenty years were comprehensively searched. Animal model, tumor type, feeding regimen, study length, sample size, major outcome, conclusion, quality assessment score and the interferential step of cancer were extracted from each eligible study. We analyzed the tumor incidence rates from 21 studies about caloric restriction. Fifty-nine studies were involved in our system review. The involved studies explored roles of dietary restriction during initiation, progression and metastasis of cancer. About 90.9% of the relevant studies showed that caloric restriction plays an anti-cancer role, with the pooled OR (95%CI) of 0.20 (0.12, 0.34) relative to controls. Ketogenic diet was also positively associated with cancer, which was indicated by eight of the nine studies. However, 37.5% of the related studies obtained a negative conclusion that intermittent fasting was not significantly preventive against cancer. Caloric restriction and ketogenic diet are effective against cancer in animal experiments while the role of intermittent fasting is doubtful and still needs exploration. More clinical experiments are needed and more suitable patterns for humans should be investigated.
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Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo. Citation: Zhuang Y, Chan DK, Haugrud AB, Miskimins WK (2014) Mechanisms by Which Low Glucose Enhances the Cytotoxicity of Metformin to Cancer Cells Both In Vitro and In Vivo. Copyright: ß 2014 Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported through grant KG100497 (W.K. Miskimins) from Susan G. Komen for the Cure. Seahorse XF24 experiments were supported by COBRE award 5P20GM10358 (W.K Miskimins) from the National Institute of General Medical Sciences at the National Institutes of Health. The project was also supported in part by a COBRE pilot award (Y. Zhuang) and PHS grant 1R01CA180033-01 (W.K. Miskimins) from the National Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
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Neuroblastoma serves as a paradigm for applying tumor genomic data for determining patient prognosis and thus for treatment allocation. MYCN status, i.e., amplified vs. non-amplified, was one of the very first biomarkers in oncology to discriminate aggressive from less aggressive or even favorable clinical courses of neuroblastoma. However, MYCN amplification is by far not the only genetic change associated with unfavorable clinical courses. So called "segmental chromosomal aberrations," (SCAs) i.e., gains or losses of chromosomal fragments, can also indicate tumor aggressiveness. The clinical use of these genomic aberrations has, however, been hampered for many years by methodical and interpretational problems. Only after reaching worldwide consensus on markers, methodology, and data interpretation, information on SCAs has recently been implemented in clinical studies. Now, a number of collaborative studies within COG, GPOH, and SIOPEN use genomic information to stratify therapy for patients with localized and metastatic disease. Recently, new types of DNA based aberrations influencing the clinical behavior of neuroblastomas have been described. Deletions or mutations of genes like ATRX and a phenomenon referred to as "chromothripsis" are all assumed to correlate with an unfavorable clinical behavior. However, these genomic aberrations need to be scrutinized in larger studies applying the most appropriate techniques. Single nucleotide polymorphism arrays have proven successful in deciphering genomic aberrations of cancer cells; these techniques, however, are usually not applied in the daily routine. Here, we present an ultra-high density (UHD) SNParray technique which is, because of its high specificity and sensitivity and the combined copy number and allele information, highly appropriate for the genomic diagnosis of neuroblastoma and other malignancies.