Mutagenesis. 2014 (May); 29 (3): 177–187 ~ FULL TEXT
Vadim Aksenov, Douglas Boreham1 and C. David Rollo
Department of Biology and Department of Medical Physics and Applied
1280 Main St. W., Hamilton,
Ontario L8S 4K1, Canada
A complex dietary supplement designed to impact multiple mechanisms associated with aging and cancer reduced overall tumorigenesis in cancer-prone heterozygous Trp53+/- mice by ~30% (P < 0.018). Carcinomas were reduced by 67% (P < 0.006). Remarkably, metastasis (a leading cause of cancer mortality) was undetectable in treated animals (P < 0.004), and the occurrence of multiple primary tumours was reduced by 74% (P < 0.012). Reduction of pulmonary adenocarcinoma by 62% (P < 0.021) was of particular note given that lung cancer is the second leading cause of death in humans. Tumours showed pronounced age-related expression in untreated animals older than 600 days. Benefits of treatment only emerged in these later ages, suggesting that the supplement acted on mechanisms common to aging and cancer. The supplement was administered daily on bagel bits that were usually eaten within minutes by the mice. Although longevity was not statistically different between treatments, longevity was strongly related to the compliance of mice in eating the supplement. Linear regression revealed a strong positive relationship between the proportion of supplement eaten and the longevity of mice within the treatment group (P < 0.0001).
From the FULL TEXT Article:
The p53 pathway crucially protects vertebrates from cancer.[1–3] Some 50–80% of human tumours are linked to loss of p53
function. [1-6] The p53 protein permits cell cycle progression
under favourable conditions, induces cell cycle arrest if repair
is required or initiates apoptosis if damage is severe. [3, 6]
Signalling by p53 responds to diverse stressors including
nutrient deprivation, hypoxia, oncogene activation and DNA
damage. [3, 6] Disruption of p53 allows damaged cells to
proliferate and progress to transformation. [1, 3] An important
role for p53 in cell senescence highlights a trade-off between
tumour suppression and aging. [1, 3, 6–8]
Mice with defective p53 are useful models for cancer studies.
Tumour suppression depends on p53 expression, and consequently,
mice with total p53 disruption have more severe
tumorigenesis than those haploid for one functional p53 gene. [2, 3, 5, 7] The lifespan of knockout (Trp53–/–) mice is drastically
reduced as they succumb to cancers within 10 months
(2). Alternatively, Trp53+/– heterozygous mice exhibit delayed
tumorigenesis manifesting at 1–2 years (and some escape
cancer).  Thus, maximal longevity of Trp53+/– mice can be
comparable to controls (~3 years) although mean survivorship
is typically reduced as ~90% of Trp53+/– mice die by 2 years
of age.  Heterozygous Trp53+/– mice are a better model for
testing cancer interventions than Trp53–/– mice since severe
tumorigenesis may overwhelm potential treatments and limit
expression of cancers to only the earliest lethal forms. Delayed
cancer in Trp53+/– mice may better resolve longer term treatment
effects on a greater range of cancer types.
Cancer is the second leading cause of death in industrialised
nations. [9, 10] Over 1.6 million new cases and over half a million
cancer deaths were projected in the USA for 2012 alone.  The probability of developing cancer strongly increases
with age with ~80% of cancer-related mortality occurring in
those over 60 years old.  We found that cancer was also
much greater in older cohorts of Trp53+/– mice indicating agerelated
trends comparable to humans. Further, there is substantial
overlap in the types of cancers in humans and Trp53+/– mice.
The four cancers commonly affecting Trp53+/– mice are lymphomas,
soft-tissue sarcomas, osteosarcomas and lung carcinomas. [2, 7] Lymphomas and lung carcinomas are among the
10 most frequent human cancers, and soft-tissue sarcomas are
relatively common (10). One exception is osteosarcoma, which
is frequent in Trp53+/– mice but rare in aging humans. Given
the pervasive dysregulation of p53 in human and murine cancers,
the similarity of cancer types and comparable age-related
trends, Trp53+/– mice provide reasonable models for general
understanding of cancer. Although humans are intrinsically
more resistant to cancer than inbred mice, benefits ameliorating
fundamental mechanisms like p53+/– may translate to humans.
Consideration of dietary supplements (DSPs) for preventing
or treating cancer grows exponentially. Searching Google
Scholar for ‘dietary supplements + cancer + prevention’ returns
3,500 articles for 2012 alone. The possible effectiveness of
nutraceuticals (particularly antioxidants) is highly controversial. [11, 12] Many studies find little benefit although a recent
clinical trial found definitive evidence for amelioration of
cancer by a commercial multiple vitamin.  Another study
found that participants ingesting high amounts of vitamins E
and C, selenium and zinc were 67% less likely to develop pancreatic
cancer.  Inconsistent results may reflect variable
experimental designs (particularly the length of treatment and
follow-up periods), diverse confounding factors, potential prooxidant
properties of antioxidants and lumping DSPs into a single
category despite enormous functional diversity. Against this
background, meta-analyses may paint effective treatments with
the stigma of majority failure. Furthermore, in vitro findings
often fail to translate to whole organisms.
Here, we show that a complex DSP designed to target multiple
mechanisms associated with aging and cancer significantly
reduced primary and metastatic tumorigenesis in Trp53+/– mice.
Effectiveness likely reflects a design targeted to multiple mechanisms
and synergistic, complementary and emergent interactions
The DSP employed here was based on our original formulation
designed to target five key mechanisms of aging. This modestly
extended longevity and strongly ameliorated age-related
declines in cognition, sensory systems and motor functions in
mice. [15–19] Given the strong age-related aetiology of many
cancers [3, 6, 10], we hypothesised that a well-designed combination
of ingredients might also impact cancer development.
Consequently, we expanded our original formulation  to
include five new ingredients relevant to tumorigenesis (Table I). Here, we report significant impacts of this DSP on diverse
tumour types and explore likely mechanisms.
Materials and methods
A total of 114 heterozygous p53 (Trp53+/–) male mice were studied (F1 progeny
of Jackson Trp53+/– males x SVJ129 +/+ females). Genotype was confirmed
by Mouse Genotype LLC, Carlsbad, CA. All protocols adhered to McMaster
University and the Canadian Council for Animal Care standards. Mice were
housed three per cage in specific pathogen-free conditions. Aggression
between cage-mates was rare, and aggressive mice were separated. Standard
rodent chow (Teklad® 8640) and water were provided ad libitum. Facilities
were maintained at 23°C ± 2°C with a 12:12 h light:dark cycle.
Complex DSP and treatment protocol
Table I lists components and dosages of the complex DSP. A review of the
properties of all 35 ingredients relevant to cancer is precluded here. The original
ingredients were targeted to offset oxidative stress, insulin resistance and
inflammation while promoting mitochondrial function and membrane integrity. [17–19] These are all variously relevant to cancer.  We added five
ingredients highlighted for their anti-cancer impacts (curcumin, resveratrol,
lycopene, quercetin and pomegranate).  Dosages were calculated from
high-end recommendations for humans adjusted for the smaller size and higher
metabolism of mice. 
Curcumin activates p53 and can inhibit proliferation, metastasis and angiogenesis
of cancers. [22–24] It has antioxidant and anti-inflammatory activity
and inhibits the cell cycle, metalloproteinases and multiple growth factors via
inhibition of signal transducer and activator of transcription (STAT) signalling. [22, 23, 25] Curcumin also inhibits activator protein 1 and promotes apoptosis
via inhibition of nuclear factor-κB (NF-κB). [23, 26] Resveratrol impacts are
similar to curcumin (antioxidant protection, p53 and apoptotic activation, inhibition
of STAT growth pathways and NF-κB). It also enhances insulin sensitivity. [24,27–30]Lycopene is an antioxidant that inhibits growth factors, NF-κB
and promotes apoptosis. [21, 31–33] Quercetin has antioxidant properties and
inhibits the cell cycle and metalloproteinases. It inhibits growth factors including
epidermal growth factor crucial to many cancers and promotes apoptosis
via activation of FOXO1. [34–37] Pomegranate can induce apoptosis in cancer
cells via inhibition of insulin-like growth factor-1 (IGF-1) signalling (and other
growth factors) highlighted in cancers. It also has antioxidant and anti-inflammatory
properties and inhibits NF-κB. [38–40]
Of the 114 mice, 60 were randomly assigned to the DSP-supplemented
group and 54 remained untreated. In previous studies, we began treatment
at weaning [15–19], but here, mice were obtained at ages between 75 and
115 days (mean age = 98 days) and supplementation was initiated immediately.
The DSP (see below) was prepared by mixing all ingredients with distilled
water and pipetting the combined slurry onto a small bread (bagel) bit (1 × 1 ×
0.5 cm). Supplemented mice received one dose each day in their home cages.
Untreated mice received just a bagel bit of standard size.
Some mice were initially reluctant to eat the entire bagel square and full
dose. Therefore, we began by feeding mice a plain bagel square (which they
consumed avidly) followed by gradually increasing the amount of DSP soaked
onto the bagel square over a 3-month period. As a result, supplementation with
a full dose was not achieved until ages of 165–205 days (~6 months). All mice
were then maintained until endpoint.
Dietary consumption index
Despite gradual acclimation, some mice did not consistently eat the entire dose.
Non-compliant mice were housed separately, so their consumption could be
monitored. We also periodically isolated other mice to obtain a consumption
index for all mice. We checked for uneaten DSP remnants immediately following
each feeding session (most mice avidly consume the entire bagel bit and
DSP within a few minutes). The weight of any uneaten piece was subtracted
from that of the initial full square. We compiled a consumption index for each
mouse as a score between 1 and 0 (where 1 indicates 100% consumption, 0.75
indicates 75% consumption, etc.). We applied regression analysis to determine
the relationship between compliance and longevity.
Longevity of control and treated Trp53+/– mice was assessed by analysing
lifetime survivorship curves. Animals found dead or culled due to tumour
endpoints were included in the survivorship analysis. In eight young treated
animals and two untreated mice, endpoint was a severe ulcerative dermatitis.
This reflects sporadic autoimmunity characteristic of C57BL/6 mice that is
unrelated to cancer.  These were excluded from the survivorship analysis.
Mice were periodically weighed between 75 and 500 days of age. Each mouse
was weighed on 10 occasions unless it died prior to 500 days. Growth rate was
assessed via regression analysis of mass against age for mice <300 days old.
Body mass plateaued at ages >400 days, and we compared this mature body
size between treatments with a t-test.
Mice were sacrificed at endpoints that included development of a large tumour,
limb paralysis, poor body condition, immobility, frailty, irregular breathing and
prolapses. Of the 114 mice, 81 were suitable for complete necropsy and histological
assessment. Five mice that were not necropsied but that expressed large
abdominal tumours (characteristic of soft-tissue sarcoma) were included in the
‘total cancer’ analysis.
Standard tissues (sternum, thymus, heart, lungs, liver, spleen, kidneys,
adrenals, thoracic spine, lumbar spine and brain) and any abnormalities
(tumours, enlarged organs, lymph nodes, etc.) were collected. All tissues were
fixed in 10% buffered formalin. Vertebrae and other mineralised tissues were
further processed in an EDTA (145 g/l) solution.
Trimmed fixed tissue sections were embedded in paraffin. Paraffin blocks
were sectioned on a Leica RM 2165 microtome at 3-µm thickness and stained
with hematoxylin and eosin for histological examination. The presence of
cancer, tumour types or other disease was diagnosed by an expert veterinary
pathologist (Dr Dean Percy, Guelph, ON, Canada) based on slide examination
and consultation of necropsy reports. Blind repeat samples were resubmitted
for quality assurance with a 100% demonstrated precision record. Full pathology
reports were obtained for 38 of 54 untreated mice and 43 of 60 supplemented
mice. Since the proportion of animals that we failed to collect tissues
from was virtually identical between treatments and causes were random, the
untested animals did not introduce any experimental bias.
Total cancers in supplemented and untreated mice and each specific type of
tumour were compared using a two-tailed chi-square (Fisher’s) test. Analysis of
tumour types was based strictly on mice that had complete necropsy examination.
The percent reduction of tumorigenesis for each cancer type was calculated
Percent reduction = 100 – (Treated/Untreated),
where ‘treated’ is percent occurrence in treated animals and ‘untreated’ is percent
occurrence in untreated mice.
Differences in survivorship between treatments were analysed with the logrank
test. Growth rate was compared with regression (homogeneity of slopes)
analysis. Mature body mass was compared using a t-test. Regression analysis
was used to test the correlation between longevity and the DSP consumption
index. All analyses were performed using Statistica® 6.0 software.
The number of mice succumbing to tumours was significantly
reduced by the DSP. Of 41 control mice, 34 (83%) expressed
tumours compared with only 26 of 45 supplemented mice
(58%) (30% reduction, P < 0.018, Table II). The predominant
tumours were sarcomas, lymphomas and carcinomas. Tumour
incidences with respect to treatments are shown in Table II.
Occurrence of sarcomas was most frequent, affecting 44% of
animals. Osteosarcoma was found in 13 untreated mice (34%)
and 8 supplemented mice (19%). The 44% reduction in incidence
of osteosarcomas in supplemented mice fell short of significance
(P < 0.132, Table II). Osteosarcomas were predominantly
observed in lumbar vertebrae, but occasionally, the skull, jaw,
liver, lung and sternum were affected. Soft-tissue sarcomas were
observed in 16% of mice (22% if the five mice that were not
necropsied are included). Of necropsied mice, hemangiosarcoma
affected three untreated and five supplemented mice. Two cases
of fibrosarcoma were diagnosed in supplemented mice and one
case in an untreated mouse. One case of histocytic sarcoma
affecting seven tissues was found in an untreated mouse. One
case of astrosarcoma (brain) and one case of chondrosarcoma
occurred in supplemented mice. No significant differences were
obtained in any of these comparisons.
Carcinoma affected 27% of animals and was diagnosed in 16
untreated and 6 supplemented mice (Table II). DSP treatment
resulted in a 67% decrease in carcinoma (P = 0.006, Table
II). Other than two cases of basosquamous cell carcinoma in
untreated mice, the rest were pulmonary adenocarcinomas.
Frequency of pulmonary adenocarcinomas was 37% in untreated
mice (14 of 38) compared with only 14% in supplemented
mice (6 of 43). The 62% reduction was statistically significant
(P = 0.021; Table II).
Lymphomas occurred in 17% of animals, affecting five
supplemented and nine untreated mice (Table II). Due to sample
size, this 50% reduction was not statistically significant. In all but
one case, lymphomas were extra-nodal affecting a wide range of
tissues, including lung, liver, spleen, kidneys and vertebrae. Often,
multiple tissues and organs were affected in the same animal.
Two cases of pheochromocytoma were diagnosed in untreated
mice. Although this tumour was not found in supplemented
animals, low incidence precluded statistical significance.
Multiple tumour burden
Multiple tumour types of primary origin were found in 13
untreated mice (34%), most frequently a combination of
pulmonary adenocarcinoma and osteosarcoma. In two cases,
three primary tumour types were present. Conversely, only
four supplemented mice (9%) had multiple tumours (Table
II). A chi-square test confirmed that the incidence of multiple
primary tumours was significantly lower in supplemented
animals (P < 0.012; Table II).
Metastases were confirmed in seven untreated mice (18%).
Remarkably, tumours of metastatic origin were absent in
supplemented animals. This difference was highly significant
(P < 0.004, Table II), indicating that the DSP effectively limited
Relationship between tumorigenesis and age
Mice sacrificed due to tumour-related endpoints were
categorised into 50-day intervals, and age-related tumour
burden is presented in Figure 1. The youngest mouse to develop
a tumour was 249 days old. Between 350 and 600 days of age,
roughly 2–6 mice were diagnosed with a tumour every 50 days.
Tumorigenesis was similar for supplemented and untreated
mice prior to 600 days of age (Figure 1) but rose steeply in
untreated mice >600 days old compared with little increase
in supplemented mice (Figure 1). Thus, benefits of the DSP
mainly manifested in older ages.
Survivorship and dietary supplementation
Surprisingly, log-likelihood analysis did not detect a significant
difference in average survivorship between DSP and control
mice (Figure 2) despite the marked reduction in lifetime
tumour frequency. This likely reflected that there was little
difference in tumorigenesis between treatments prior to
600 days with large impacts of the DSP emerging only in the
oldest age cohorts. Several untreated mice that fortuitously did
not develop cancer lived the longest (but maximal longevity did
not differ significantly for a t-test of the last 10% of surviving
mice). Eight younger supplemented and two untreated mice
were terminated as they developed ulcerative skin lesions. Such
lesions are common and specific to the C57BL/6 mouse strain.
They reflect sporadic autoimmune disease unrelated to cancer.  Regardless, longevity was strongly linked to rates of DSP
consumption. Two mice had a DSP consumption index of <0.6;
85% of mice had an index of 0.8 or better; and 62% and 41%
had indices of >0.9 or >0.95, respectively. Plotting the longevity
for each supplemented mouse against its consumption index
revealed a strong positive relationship between longevity and
consumption of the DSP (P < 0.0001, Figure 3). The longest
lived mice were those consistently ingesting the highest doses,
whereas the shortest lived mice ate the least DSP (Figure 3).
We regularly recorded body mass from 90 to 500 days of age.
Figure 4A illustrates that the growth rate of mice <300 days
of age was significantly higher in untreated compared with
supplemented mice (linear regression, homogeneity of slopes
analysis, P < 0.002). Comparison of mean adult body mass
between treatments (mice >400 days old) with a t-test was
not statistically significant (P > 0.25, Figure 4B). Despite
significantly slower growth in DSP mice, body mass between
the treatment groups at 290 days differed by only 3.6%.
Overview of tumorigenesis
The DSP reduced the number of Trp53+/– mice succumbing to
tumours by ~30% (P < 0.018, n = 86; Table II). The biggest
reduction (67%) was in carcinoma (P < 0.006). Despite clear
trends for other cancers, low sample sizes obscured statistical
significance. For example, lymphomas affected only 17% of
mice, so despite a 50% reduction in treated mice, impacts of
the DSP proved not significant (Table II). Pheochromocytomas
occurred exclusively in untreated mice but only two cases
were diagnosed (Table II). Osteosarcomas were found in 26%
of mice, but despite being 44% less frequent in supplemented
mice, this also fell short of significance (P = 0.132; Table II).
A third of untreated Trp53+/– mice expressed multiple primary
tumours (usually adenocarcinoma and osteosarcoma)
(Table II). Conversely, multiple primary tumours afflicted only
9% of supplemented mice (a reduction of 74%, P < 0.013; Table
II), strongly supporting effectiveness of the DSP against tumorigenesis.
This may be particularly relevant to therapeutic applications
where treatment of multiple tumours is challenging.
Cancer types in Trp53+/– versus normal mice
Sarcomas, lymphomas and carcinomas collectively comprise
>95% of tumours occurring in mice. [2, 7] However, Trp53+/–
mice exhibit significantly different proportions of tumours
compared with normal mice. Thus, sarcomas typically
comprise 20% of tumours in normal controls but can exceed
60% in Trp53+/– mice. [2, 7] We did not examine normal mice
but we did find a high frequency of sarcomas (50%) in untreated
Trp53+/– mice (Table II). In general, lymphoma and carcinomas
are only 2- to 4-fold higher in Trp53+/– mice compared with
normal controls (i.e. loss of p53 has a relatively small impact on
frequency of these tumours), whereas sarcomas can be elevated
15- to 30-fold. 
Despite a hopeful trend for osteosarcoma,
sarcomas generally were unresponsive to supplementation
(20% reduction by the DSP, P = 0.378). Our overall estimate of
effectiveness of the DSP (~30%) included sarcomas. Excluding
sarcomas, the frequency of all other cancers in mice amounted
to 71% of untreated mice, whereas DSP treatment reduced this
to 26% (a 40% reduction, P < 0.0002). Considering that normal
animals exhibit far fewer sarcomas and more carcinomas and
lymphomas (that responded better to the DSP), DSP benefits
would likely be relatively greater in normal mice with intact
p53. In humans, sarcomas are also less frequent than lymphomas
and carcinomas are similarly resistant to treatment. 
Lung cancer comprised 14% of cancers diagnosed in the USA
in 2012 but accounted for ~29% of cancer deaths, as lung cancer
is refractive to treatment.  Pulmonary adenocarcinoma
is the most frequent lung cancer worldwide and rates are
increasing.  Remarkably, the DSP obtained 62% reduction
in pulmonary adenocarcinoma in mice (P < 0.021, Table
II). This is one of our most promising results given the high
morbidity of this disease. Even modest translation to humans
would yield large economic and social benefits.
Suppression of metastasis by the DSP (Table II, P < 0.004) is
particularly important given that radiation or chemotherapy is
required for treatment [43, 44] and malignancy contributes to
90% of cancer deaths.  Several aspects of cell transformation
and angiogenesis are regulated by redox, suggesting an
antioxidant mechanism for our results.  Likely contributors
include matrix metalloproteinases (MMPs) and growth factors,
such as IGF-1 [47–49], transforming growth factor-β and
vascular endothelial growth factor (VEGF). [46,50,51] All are
favoured by oxidative conditions. [46, 50, 52, 53] Inflammatory
cytokines also contribute to MMP expression  and stimulate
NF-κB, STAT  and VEGF.  Nearly one-third of cancer
patients exhibit metastasis at first diagnosis. 
Cancers obtain high evolvability via an elevated oxidative milieu
and strong survival signal. This facilitates adaptation to the host
and treatments via complex alterations in cellular regulatory networks.
Strong protection against radiation-induced DNA damage  by our original DSP suggests that mutagenesis essential for
cancer progression and metastasis may be significantly reduced.
Indeed accelerated growth of tumour cells with p53 deficiency was
suggested to reflect increased mutagenesis and malignant progression.  Inhibition of metastasis by the DSP could extend the
period for effective surgical intervention.  Interestingly, aspirin
(in the DSP) reduces metastasis in humans. 
Aging and cancer
Cancers in untreated Trp53+/– mice showed strong age-related
expression, whereas the increase in mice older than 600 days
was much less in treated animals (Figure 1). The original DSP
was designed to modulate five key mechanisms associated with
aging (reduce free radical processes, ameliorate inflammation,
increase insulin sensitivity, improve mitochondrial function
and maintain membrane fluidity). [15–18] Age is the primary
risk factor for many cancers [2, 10], and success of the DSP
likely involves mechanisms common to both aging and cancer. [1, 3, 6] Full-dose supplementation was only achieved by
~6 months of age when some tumorigenesis could have been
initiated. Better results may have been obtained with earlier
intervention. Tumours take time to attain detectable size ,
and some mice reached endpoint at 250–350 days of age.
Regardless, treatment benefits were most apparent in older ages
that represent the most relevant cohort for assessing impacts.
Mutation accumulation with age is undoubtedly important, but
aging of the organismal/cellular environment [e.g. signalling,
redox, senescent cells, functional declines (proteasome,
autophagy, DNA repair)] could also impact cancer. 
Oxidative stress, the genome, cellular damage and tumorigenesis
The free radical theory of aging faces challenge, but there is a
clear causal role of oxidative stress in cancer. Most spontaneous
tumours trace to DNA damage and mutations driven by genotoxic
stress (particularly reactive oxygen and nitrogen species
[ROS]). [1, 3, 6, 45, 63–66] Several antioxidants and DNA repair
mechanisms are regulated by p53. [59, 67] If DNA damage is
detected, p53 arrests the cell cycle and promotes repair. If damage
is severe, p53 promotes apoptosis or transition of cells to a
senescent phenotype. [1, 3, 6] Reduction of DNA damage is likely
to ameliorate cancer [31, 64, 68, 69], whereas low manganese
superoxide dismutase (MnSOD) activity promotes cancer. 
Although the status of specific antioxidant enzymes and
redox strongly vary among cancers and their stage of development,
imbalanced antioxidant function and an oxidative
milieu characterise most cancers. [71, 72] Overexpression of
mitochondrial catalase ameliorated breast cancer in mice ,
including metastasis as in our study. N-acetylcysteine also
strongly ameliorated cancer in Trp53–/– knockout mice.  Of
the 35 ingredients in our DSP (including N-acetylcysteine), 27
scavenge diverse mitochondrial and cytoplasmic ROS, including
H2O2, O2–, OH–, NO, NO–, O– and ONOO– . [17–19,58]
Chromosomal aberrations and oxidative DNA base damage
are associated with cancer [63, 64, 74], and our original DSP
prevented damage by 2 Gy of whole body ionising radiation
(likely by rapid scavenging of radiation-induced free radicals).
The DSP also reduced radiation-mediated apoptosis of
lymphocytes, likely by preventing damage.  We further
showed that oxidative and nitrosative protein damage in brains
of supplemented mice were significantly reduced [17, 19],
whereas oxidised and nitrated proteins are elevated in cancers.  A multiple DSP (23 vitamins, minerals and antioxidants)
reduced oxidative DNA damage in lymphocytes of humans
45–70 years old , suggesting promise for our DSP.
Some debate regarding the free radical theory of aging
reflects that some radicals are essential signalling molecules,
whereas others, such as peroxynitrite, are highly damaging.
Our original DSP ameliorates nitrosative stress even though
this was not a design target.  Strong antioxidant activity of
the DSP could ameliorate DNA damage, mutations and tumorigenesis
across the lifetime of mice.  Aging and cancer may
also reflect oxidative impacts on aspects such as mitochondrial
integrity and biogenesis, proteasome function and autophagy.
The DSP could protect these and redox-regulated ion channels
supporting cellular functions. [77, 78]
Normally, superoxide generated by mitochondria is removed
by MnSOD.  However, age-related accumulation of damage
can impact electron transport chain (ETC) complexes [17, 18, 80, 81], leading to reduced energy and increased ROS
generation.  Thus, maintaining mitochondrial function
could reduce oxidative stress, DNA damage and mutagenesis.
Several DSP ingredients support mitochondrial function ,
and our original DSP increased activity of mitochondrial ETC
complexes II–IV and reduced oxidative damage. [17–19]
Long-lived calorically restricted (CR) mice express low levels
of ROS [82, 83] via decreased mitochondrial ROS generation. [82–84] This may contribute to reduced tumorigenesis. 
The DSP mimics some aspects of CR with respect to mitochondria [17–19] implicating improved mitochondrial function in
the amelioration of cancer achieved here.
Inflammation and cancer
Inflammation was a design criterion for the original DSP  and is implicated in both aging and cancer. [86–88]
Inflammation can impair antioxidant defenses  and
increase ROS stress by activating NAD(P)H oxidases, nitric
oxide synthase, myeloperoxidase, eosinophil peroxidase 
and 5-lipoxygenase.  Nicotinamide adenine dinucleotide
phosphate oxidase (in macrophages and neutrophils) and
5-lipoxygenase (in lymphocytes) are activated by inflammatory
cytokines such as interleukin-1β  and can generate
large amounts of ROS.  This can in turn induce further
inflammatory cytokines.  Inflammation-mediated ROS
can exacerbate mutagenesis and cancer.  Interleukin-6 is
particularly important in cancer as it can promote proliferation
and anti-apoptotic protection to cells expressing its receptor.  Anti-inflammatory drugs protect against cancer. [91–94]Hence, anti-inflammatory agents (including aspirin
and antioxidants) in the DSP may contribute to reduced
tumorigenesis in Trp53+/– mice (Table II).
Insulin resistance and tumorigenesis
Insulin resistance commonly develops with age [95, 96] and
contributes to age-related pathologies including cancer. [97–99]
There are 11 ingredients in our DSP that promote insulin sensitivity , and the original DSP lowers blood glucose and improves
glucose clearance (S. Matravadia and V. Aksenov, unpublished
theses). The mechanisms linking insulin resistance to cancer are
unclear, but the anti-cancer impacts of aspirin, the diabetes drug
metformin and resveratrol likely involve upregulation of the key
energy sensor, AMP-activated protein kinase (AMPK). [100, 101]
Signals of low energy (activated AMPK) can inhibit target of
rapamycin (TOR) and upregulate FOXO. [102, 103] Although
such actions can improve mitochondrial function , our
DSP appears to increase energy supply. The DSP may act via
diverse mechanisms such as increasing spontaneous exercise  and elevated stress-resistance and repair mechanisms (e.g.
proteasome activity, antioxidant recharging and DNA repair).
Longevity, cancer and supplementation
Survivorship curves did not differ significantly between
supplemented and control mice (Figure 2). Given that DSP
impacts were largely restricted to mice >600 days old and the
log-rank test is a measure of general health across the lifespan,
this may not be surprising. Also, the lack of significant extension
of maximal lifespan does not reflect negative consequences of
the DSP since mice showed a strong positive relationship of
longevity with compliance, and untreated Trp53+/– mice that
escaped cancer can achieve normal life spans (Figure 2). Aging
and cell senescence are regulated by p53 [3, 6, 7, 104] which may
could account for lack of longevity extension by the DSP in this
strain, whereas a small increase in longevity was previously
obtained in normal outcrossed mice (Figure 2). 
DSP compliance and survivorship
Some mice did not consume a full dose of DSP on a daily basis.
Forty-one percent of mice consistently ingested >95% of their
daily dose and >85% of mice ingested at least 80% of the full dose
over their lifetime. Previous studies with our original DSP obtained
full compliance. [15–19] Greater bulk and taste of the cancer DSP
likely impacted compliance. Most early deaths of supplemented
mice were those showing poor compliance for ingesting the
supplement. Thus, of six mice with a consumption index <0.78,
none lived longer than 400 days compared with >700 days for some
mice with compliance ratios close to 1.00 (Figure 3). As suggested
by a reviewer, we re-examined the relationship between longevity
and consumption index with the six poor eaters removed (<0.78
index). The regression for the remaining mice remained highly
significant (P < 0.005) confirming that the relationship reported
in Figure 3 was not artificially inflated by outlying points. Given
that cancer is the main determinant of longevity for this strain, the
significant benefit of the DSP on survivorship of Trp53+/– mice
reinforces the anti-cancer actions of the diet.
Body size, supplementation and p53
The p53 pathway negatively impacts growth and proliferation
via engaging cell cycle arrest.  Mice with amplified
p53 expression have reduced body weight and diminished
tumorigenesis (i.e. body mass is negatively associated with
p53 expression). [7, 104] Remarkably, the size range for male
C57BL/6 mice (Jackson Laboratory Phenotype Information) is
generally <35 g, but our Trp53+/– mice attained mature sizes of
>48 g (Figure 4). The DSP had a significant but small impact
on growth rates, but no change in adult mass was obtained
(Figure 4). This suggests that the DSP did not act via a dietary
restriction mechanism. Curcumin and resveratrol (both in our
DSP) can increase p53 expression and nuclear translocation  suggesting that the reduction in early growth rate could
reflect p53 impacts.
Impacts on growth, however, were small
and could just as well reflect reduced oxidative conditions.
Activity of growth-promoting pathways (e.g. growth harmone
and TOR) is associated with elevated ROS [102, 103], and
antioxidants in the DSP could inhibit such signalling. We
obtained very similar reduction in growth rates (with no
change in mature size) in normal C57/BL6-SJL mice fed our
original complex supplement rich in antioxidants. In that case,
there was a significant 11% extension in life span  but no
reduction in food intake relative to untreated controls (P = 0.95,
unpublished). The original DSP also increased motor activity
of mice  that could also reduce growth efficiency. It is also
worth mentioning that the maximal longevity of ~800 days
in Trp53+/– mice that escaped cancer is not atypical of the
C57BL/6 strain. 
The size of Trp53+/– mice approaches that of transgenic
growth hormone mice (Tg). We previously obtained a statistically
significant increase in longevity of Tg mice (27%) with
our original DSP.  Various types of Tg also have increased
susceptibility to cancer (Prof. A. Bartke, personal communication)
as might be expected from the known impacts of
growth hormone and IGF-1 on the TOR pathway and cancer. [49, 102, 103] Some increase in the survival of supplemented
Tg likely reflected reduced cancers.
Clinical potential of the DSP
An 11-year study of treatment and follow-up for cancer in >14
000 physicians taking a daily multivitamin (Centrum Silver®)
(versus placebo) found modest but significant reductions in
total cancer with no indicators of significant harm.  This
is one of the first clinical studies to experimentally address a
truly broad-spectrum formulation. Overall, a 6.36% reduction
in total cancer was observed. Excluding prostate cancer, the
overall reduction was ~11%. For those with a baseline history
of cancer, treatment reduced total cancer by 22.6%.
This study involved men 50 or older. Examination of cancer
incidence with greater follow-up time showed progressively
greater divergence (improved resolution) between treated and
untreated groups for cumulative cancer and colorectal cancer
(but not prostate cancer). Similar trends for some cancers
were reported by others. [106, 107] Given the likely long-term
cumulative benefits of supplements and prolonged age-related
development of tumorigenesis, substantial monitoring and follow-
up periods are required to detect cancer benefits in humans. [13, 108] Our lifetime study of mice is instructive in that
expression of cancers and protection by the DSP were largely
limited to advanced ages (Figure 1).
Many studies testing individual vitamins and some testing
multivitamins have detected no benefit for cancer. 
Alternatively, extensive evidence supports effectiveness of
diverse phytochemicals on pathways relevant to aging and cancer.  Given the well-documented pro-oxidant capacity of
many antioxidants when given individually in high doses, negative
results can be a prophecy of design that for clinical trials
seems ill advised. Many negative studies classified as ‘multivitamin’
test only 2–4 ingredients, so pro-oxidant risks likely
remain. Prevalence of such studies and those of insufficient
duration must strongly bias outcomes of meta-analyses where a
truly effective formulation could be averaged out.
Despite considerable evidence against nutraceutical efficacy
against cancer, other studies support the Centrum® multivitamin
trial.  The ‘Linxian Chinese Cancer Prevention Trial’
found a 13% reduction in cancer mortality and a 9% reduction
in total mortality for a supplement containing beta carotene,
vitamin E and selenium.  Remarkably, benefits of multivitamin
supplementation persisted for 10 years following completion
of the trial, although this was largely limited to participants
younger than 55 years.  A study testing a combination of
vitamin C, vitamin E, beta carotene, selenium and zinc obtained
significant benefit for cancer and total mortality limited to men
(107). The ‘Nurses’ Health Study’ detected that prolonged use
of multivitamins with folate ameliorated colon cancer but only
in a 15-year follow up.  The ‘Cancer Prevention Study II’
found an inverse relationship between long-term multivitamin
use with colon cancer and mortality.  A study of vitamin
E and C, selenium and zinc intake in >23,000 participants who
are 40–74 years old found that those with diets containing high
amounts were 67% less likely to develop pancreatic cancer. 
Another large study calculating the total antioxidant capacity of
an Italian diet found a significant inverse relationship between
colon cancer and dietary antioxidant capacity.  It must be
emphasised that the DSP is an anti-cancer cocktail and not simply
an antioxidant supplement. In this regard, the efficacy of
aspirin in reducing diverse cancers (~20% reduction overall) is
established for humans. [93, 94]
Our study differs from others in the larger number and higher
dosages of ingredients employed, as well as a formulation
designed to impact multiple mechanisms of aging and cancer
(i.e. all supplements are not equal and the whole is greater than
the sum of the parts). This may simultaneously obtain global
alterations in redox and energy status and diverse impacts on
cellular regulatory circuitry. This may be particularly relevant
to cancers that exploit multiple pathways to evolve greater
oncogenic potential and resist treatments.
Potential mechanisms of DSP action
Mechanisms explaining amelioration of cancer by the DSP
(i) reduction of ROS and prevention of genotoxic stress (suppressing initiation of cancer, regulatory distortion and metastatic evolution);
(ii) growth and cell cycle inhibition and
(iii) reducing signals inhibiting apoptosis that are generally over expressed in cancer.
We have good support for the first.
Although we found a small inhibition of growth, it seems
insufficient to explain the degree of cancer reduction. Release of
apoptosis by reducing the oxidative environment and enabling
suppressed signalling systems is highly controversial.
Upregulation of TOR and growth by oxidative conditions
can suppress FOXO. [102, 103] Many crucial enzymes involved
in control of redox, repair, the cell cycle and apoptosis contain
cysteine residues conveying redox regulation (including p53).  Thus, reducing the oxidative environment of cancer could
inhibit IGF-1 signalling and TOR (and their associated survival
signal) while upregulating pro-apoptotic FOXO. Several studies
found that antioxidants (N-acetylcysteine, tempol, propyl
gallate, dithiothreitol, melatonin) potently induce apoptosis in
some cancers including breast cancer cells with breast cancer 1
(BRCA1) deficiency and colon cancer. [114, 115] Others argue,
however, that effects of antioxidants reflect their pro-oxidant
potential.  Increasing oxidative stress is also highlighted
in chemotherapy.  The controversy particularly focuses on
the Kelch like-ECH-associated protein 1 (KEAP1)/ nuclear factor
2 (NRF2) stress response system. Although FOXO may be
inhibited in cancer, NRF2 can be activated to potentially protect
cancers from excessive oxidative stress and chemotherapy. 
Our DSP was designed to ensure recycling among antioxidants,
and we documented that our original formulation
has antioxidant rather than any pro-oxidant properties. [17, 19, 58, 75] We are currently examining apoptosis in cancers
found in DSP-treated mice to determine if cell killing might
be achieved by reducing the oxidative conditions supporting
cancer or by otherwise supporting FOXO activity.
Some suggest that phytochemicals and antioxidants have no
anti-aging or cancer benefits. Our results certainly do not support
Watson’s 2013  claim that antioxidants cause cancer,
although it is likely that the DSP could impede chemotherapy
agents that act via elevated oxidative stress. The real question is
whether the DSP itself is a novel form of chemotherapy.
C.D.R. was supported by a grant from the Natural Sciences
and Engineering Research Council of Canada and D.B. was
supported by a grant from the US Department of Energy
Low Dose Radiation Research Program (grant number
We thank Zoya Tov for assisting in preparation of the dietary supplement,
Veterinary Pathologist Dr Dean Percy for carrying out the histopathological
examinations, Prof. Joao Pedro de Magalhaes for helpful suggestions in review
and our editor Dr Charles Limoli.
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