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Ait-Ghezala—Telomerase Activation With Natural Compounds
6 ALTERNATIVE THERAPIES, VOL. 22 NO. S2
Identication of Telomerase-activating Blends
From Naturally Occurring Compounds
Ghania Ait-Ghezala, PhD; Samira Hassan, MS; Miles Tweed, MS; Daniel Paris, PhD;
Gogce Crynen, PhD; Zuchra Zakirova, MS; Stefan Crynen, PhD; Fiona Crawford, PhD
ORIGINAL RESEARCH
ABSTRACT
Context • Telomeres are repeated deoxyribonucleic acid
(DNA) sequences (TTAGGG) that are located on the
5’ ends of chromosomes, and they control the life span of
eukaryotic cells. Compelling evidence has shown that the
length of a person’s life is dictated by the limited number
of times that a human cell can divide. e enzyme
telomerase has been shown to bind to and extend the
length of telomeres. us, strategies for activating
telomerase may help maintain telomere length and, thus,
may lead to improved health during aging.
Objective • e current study intended to investigate the
eects of several natural compounds on telomerase
activity in an established cell model of telomere shortening
(ie, IMR90 cells).
Design • e research team designed an in vitro study.
Setting • e study was conducted at Roskamp Institute
in Sarasota, FL, USA.
Intervention • e tested single compounds were
(1) α-lipoic acid, (1) green tea extract,
(2) dimethylaminoethanol L-bitartrate (DMAE L-bitartrate),
(3) N-acetyl-L-cysteine hydrochloride (HCL), (4) chlorella
powder, (5) L-carnosine, (6) vitamin D3, (7) rhodiola PE
3%/1%, (8) glycine, (9) French red wine extract, (10) chia
seed extract, (11) broccoli seed extract, and (12) Astragalus
(TA-65). e compounds were tested singly and as blends.
Outcome Measures • Telomerase activity for single
compounds and blends of compounds was measured by the
TeloTAGGG telomerase polymerase chain reaction (PCR)
enzyme-linked immunosorbent assay (ELISA). e 4 most
potent blends were investigated for their eects on cancer-
cell proliferation and for their potential eects on the
cytotoxicity and antiproliferative activity of a
chemotherapeutic agent, the topoisomerase I inhibitor
topotecan. e benets of 6 population doublings (PDs)
were measured for the single compounds, and the 4 blends
were compared to 3 concentrations of eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA).
Results • Certain of the compounds increased telomerase
activity, and combinations of the top-ranking compounds
were able to increase telomerase activity signicantly,
from 51% to 290%, relative to controls.
Conclusions • e results have conrmed that many
naturally occurring compounds hold the potential to
activate telomerase and that certain of those compounds
have demonstrated synergistic eects to produce more
potent blends. Given the relationship between telomere
shortening, aging, and the decline of tissue function, it is
reasonable to hypothesize that such telomerase-activating
blends may have health-promoting benets, particularly
in relation to aging-associated conditions. Further
investigation of such blends in human studies that are
designed to evaluate safety and the eects on telomere
length are thus warranted. (Altern er Health Med.
2016;22(S2):6-14.)
Ghania Ait-Ghezala, PhD, is a scientist at the Roskamp
Institute, in Sarasota, Florida, and a research scientist at
James A. Haley Veterans Hospital in Tampa, Florida. Samira
Hassan, MS, is a research assistant; Miles Tweed, MS, is a
research assistant; and Stefan Crynen, PhD, is a scientist at
the Roskamp Institute. Daniel Paris, PhD, is a scientist at
the Roskamp Institute and a research scientist at James A.
Haley Veterans Hospital. Gogce Crynen, PhD, is a scientist
at the Roskamp Institute and a research scientist at the
Open University, Milton Keynes. Zuchra Zakirova, MS, is a
research assistant at the Roskamp Institute and a research
assistant at James A. Haley Veterans Hospital. Fiona
Crawford, PhD, is president and CEO of the Roskamp
Institute and a VA research career scientist at James A.
Haley Veterans Hospital.
Corresponding author: Ghania Ait-Ghezala, PhD
E-mail address: gaitghezala@rfdn.org
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Ait-Ghezala—Telomerase Activation With Natural Compounds ALTERNATIVE THERAPIES, VOL. 22 NO. S2 7
Telomeres are repeated deoxyribonucleic acid (DNA)
sequences (TTAGGG) located on the 5’ ends of
human chromosomes. e length of the telomere in
human somatic cells is believed to be heterogeneous, ranging
from 5 to 20 kb and is determined by a person’s age, the organ
examined, and the number of divisions that each of its cells
has endured.1
A telomere can lose up to 200 bases with each DNA
replication cycle. e progressive shortening of telomeres that
follows is one of the molecular mechanisms associated with aging,
because critically short telomeres trigger chromosome senescence
and loss of cell viability.1-3 Telomere shortening occurs as a result
of the progressive loss of these repeated nucleotide sequences that
takes place over many replication cycles. at loss can be stopped
and even reversed by the enzyme telomerase.4,5
Telomerase is a ribonucleoprotein enzyme that lengthens
the telomere by extending the DNA termini. Telomere length
has also been found to decrease with age in humans, suggesting
a role between telomere length and the fate of cells and the
decline of tissue function that eventually aects mortality.6
It h as been suggested that sh elter in, a protein complex
with DNA remodeling activity, acts together with several
associated DNA-repair factors to fold the telomere’s end into a
properly capped structure, thereby protecting chromosomes’
ends.7 It is also known that cellular senescence can be triggered
by the accumulation of too many uncapped telomeres and that
the repair of critically short telomeres by telomerase or
recombination is limited in most somatic cells. erefore, the
length of the telomere repeats can regulate the proper capping
of the ends of the chromosomes and protect them.
Telomerase aects longevity by preventing premature
telomere erosion, with that loss exemplied by human
diseases with mutations in telomerase components.
Individuals with such mutations oen develop premature
dysfunction of adult stem cells and show signs of decreased
longevity due to accelerated rates of telomere shortening.2,8-12
Although no evidence is available that telomerase drives
the oncogenic process, it is permissive and required for the
sustained growth of cancers. erefore, almost all human
cancers show telomerase activation as a hallmark process, and
it is the likely mechanism that allows unlimited cell proliferation
of tumor cells.13 However, telomere erosion has also been
documented in hyperproliferative disease states, as a condition
strongly associated with increased cancer risk. Short telomeres
have a higher predisposition to induce chromosome
rearrangements that can lead to cancer, and it has been
suggested that short telomeres may actually be a cause of
cancer14,15 and may precede reactivation of telomerase.5,16,17
Two studies have suggested that the activation of
telomerase is sucient to delay aging and increase the
lifespan in normal aging and accelerated aging mice, without
any increase in cancer incidence.18-20 Many more studies have
suggested that the reactivation of telomerase in adult or aged
organisms has a similar positive eect on the delay of aging
and that the mechanism may be independent of its role in
cancer proliferation where it is aberrantly expressed.
Given the projected increase in life span of the human
population, healthy aging might be facilitated by approaches
that maintain telomere length. A few compounds are already
commercially available, in the form of dietary supplements,
that claim to maintain or repair telomere length and that are
associated with increased longevity through telomerase
activation.18
e present study was designed to investigate the eects
of a variety of natural compounds on telomerase activity,
followed by an evaluation of whether combinations of the
most potent telomerase modulators might enhance the
performance of any single modulator. Also, given the tight link
between telomerase activity, cellular senescence, and cancer,
the eects of the 4 most eective blends for the increase of
telomerase activity were investigated regarding their eects on
tumor cell proliferation and survival in response to a
chemotherapeutic agent, to assess any potential cancer risk
associated with their promotion of telomerase activity.
METHODS
e eects of the current study’s compounds and their
combinations on telomerase activity were tested in vitro using
primary human IMR90 cells. e cells were treated with each
compound for 15 population doublings (PDs) and compared
to the commercially available telomerase activator, Astragalus
(TA-65), which has been shown to have benecial eects by
increasing telomerase activity.18,21-23 e 4 blends that showed
the highest performance were also compared in the same assay
to the eects of omega-3 fatty acids (FAs), which have been
shown to increase telomere length in a human study.24
Cells
e telomeres of IMR90 cells shorten by an average of
70 base pairs per division.25 ose cells were chosen for the
current study. IMR90 cells were purchased from ATCC
(Rockville, MD, USA) and the cells were grown in Eagle’s
minimum essential medium (EMEM) from the American
Type Culture Collection (ATCC) (Manassas, VA, USA),
supplemented with 10% fetal bovine serum in a 5% CO2,
humidied environment at 37°C.
Procedures
Single Compounds. e tested compounds were
(1) α-lipoic acid, (1) green tea extract, (2) dimethylaminoethanol
L-bitartrate (DMAE L-bitartrate), (3) N-acetyl-L-cysteine
hydrochloride (HCL), (4) Chlorella powder, (5) L-carnosine,
(6) vitamin D3, (7) rhodiola PE 3%/1%, (8) glycine,
(9) French red wine extract, (10) chia seed extract,
(11) broccoli seed extract, (12) Astragalus extract, and
(13) TA-65, which acted as the positive control. TA-65 has
been previously described as a compound capable of
increasing average telomere length by turning on the hTERT
gene, which activates telomerase.
All of the compounds tested were provided by
Enzymedica (Venice, FL, USA). Eicosapentaenoic acid
(EPA)—20:5, n-3; docosahexaenoic acid (DHA)—22:6, n-3;
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Ait-Ghezala—Telomerase Activation With Natural Compounds
8 ALTERNATIVE THERAPIES, VOL. 22 NO. S2
and delipidated and charcoal-treated fetal bovine serum were
all purchased from Sigma-Aldrich (St Louis, MO, USA).
Blends of Compounds. Eight compounds were tested:
(1) BL-1: rhodiola PE 3%/1%, sulforaphane glucosinolate
(SGS) broccoli seed extract, Astragalus extract, L- carnosine,
N-acetyl-L-cysteine HCL, and vitamin D3; (2) BL-2: rhodiola
PE 3%/1%, SGS broccoli seed extract, Astragalus extract,
L-carnosine, N-acetyl-L-cysteine HCL, vitamin D3, and an
enzyme blend containing xylanase, pectinase, hemicellulase,
cellulase TB, protease, and catalase; (3) BL-3: Astragalus
extract, SGS broccoli seed extract, rhodiola PE 3%/1%, and
vitamin D3; (4) BL-4: Astragalus extract, SGS broccoli seed
extract, and rhodiola PE 3%/1%; (5) BL-5: Chlorella powder,
Astragalus extract , rhodiola PE 3%/1%, N-acetyl-L-cysteine
HCL, SGS broccoli seed extract, L-carnosine, and vitamin D3;
(6) BL-6: rhodiola PE 3%/1%, N-acetyl-L-cysteine HCL,
L-carnosine, SGS broccoli seed extract, and vitamin D3;
(7) BL-7: Chlorella powder, glycine, and vitamin D3; and
(8) BL-8: rhodiola PE 3%/1%, L-carnosine, SGS broccoli seed
extract, L-carnosine, Chlorella powder, and TA-65.
Enzymedica currently markets BL-3 as Telomere Plus.
Lactate Dehydrogenase Assay. Regarding the cell
treatments and sample collection, all compounds were rst
tested for their toxicity by measuring the release of lactate
dehydrogenase (LDH) from Roche (Indianapolis, IN, USA) at a
dose range of 50 μg/mL to 1000 µg/mL. e cells were then
treated with each compound for 15 PDs, unless stated otherwise.
Overall, the cells were passaged into several asks and allowed
to attach for a few hours, before subsequently being treated for
72 hours with the test compound. Aer the 72 hours, the cells
were detached with TrypLE (Invitrogen, Grand Island, NY,
USA). Half of the cells were washed and ash frozen in a -80°C
freezer for analyses, and the other half were seeded in a fresh
ask and treated with the same compound for 72 hours. e
same paradigm was repeated for up to 15 PDs.
Sample Preparation. Aer the last PD treatment and
sample collection (PD-15), cells from all passages, including
previously frozen passages, were resuspended in a 150-L lysis
reagent provided in the telomeric repeat amplication protocol
(TRAP) assay, precooled on ice, and incubated for 30 minutes
on ice. e samples were prepared as per the manufacturer’s
recommendation; briey, cell lysates were centrifuged at
16
000
×
G for 20 minutes at 2°C to 8°C. e supernatant was
carefully collected, and the protein content was subsequently
quantied using a bicinchoninic acid (BCA) assay.
Outcome Measures
TRAP Assay. e telomerase activity was measured by a
TeloTAGGG telomerase polymerase chain reaction (PCR)
enzyme-linked immunosorbent assay (ELISA) assay from
Roche. It is a photometric enzyme immunoassay that is used
for the detection of telomerase activity using TRAP. e
procedure was performed in accordance with the
manufacturer’s instructions. Briey, a rst-step PCR elongation
and amplication was followed by an ELISA detection step.
For each reaction, the negative control was a heat-treated
sample, and the positive control was a human embryonic
kidney (HEK) cell extract that was provided in the kit.
Cytotoxicity and Cell Proliferation Screening. e
4 blends that were found to be most potent for telomerase
activity—BL-1, BL-2, BL-3, and BL-4—were also investigated for
their eects on cancer cell proliferation. Adenocarcinomic,
human, alveolar basal epithelial cells (A549 cells) were grown in
Dulbecco’s modied Eagle medium (DMEM) from ermoFisher
Scientic (Grand Island, NY, USA), which was supplemented
with 10% fetal bovine serum and 1× penicillin/streptomycin. In
addition, the potential impacts of BL-1, BL-2, BL-3, and BL-4 on
the cytotoxicity and the antiproliferative activity of a
chemotherapeutic agent, the topoisomerase I inhibitor topotecan,
was investigated at a dose range of 0.1 μM to 10 μM of topotecan.
Cytotoxicity was monitored by measuring the release of
LDH in the culture media, following 48 hours of treatment
with a dose range of topotecan. In addition, the number of
live cells adherent to the cell culture wells was quantied by
measuring the cleavage of the tetrazolium salt WST-1 to
formazan by cellular mitochondrial dehydrogenases
(Biovision, Milpitas, CA, USA), to determine the eect of the
dierent treatments on cellular proliferation.
Preparation of Albumin-bound FAs. Cultured IMR90
cells were incubated with albumin-bound EPA—20:5, n-3;
sigma or DHA—22:6, n-3; sigma, for 72 hours in EMEM
containing 10% delipidated and charcoal-treated, fetal bovine
serum to minimize interference from serum FAs and to
obtain a better-dened system.
Briey, the EPA and DHA were resuspended in ethanol
and stored at -20°C under nitrogen. e concentrations of
DHA and EPA to be tested were evaporated to dryness under
reduced pressure and the FAs were complexed with the
depleted bovine serum albumin in a 1:1 molar ratio according
to the method described by Mahoney.26 e n a l c o n ce n t ra t i on s
were EPA 25 μM/DHA 2.5 μM; EPA 2.5 μM/DHA 0.25 μM;
and EPA 0.25 μM/DHA 0.025 μM. e treatment was carried
out for 6 PDs and compared with the 4 most potent blends,
BL-1, BL-2, BL-3, and BL-4.
Statistical Analysis
A matched-pairs t test was used to assess signicant
changes between 2 time points. Statistical signicance was
set at α
<
.05 for all statistical analyses. Statistical signicance
is indicated with a notation in graphs.
RESULTS
LDH Assay
e results revealed that 24 hours of treatment with
200 μg/mL of DMAE L-bitartrate, N-acetyl-L-cysteine HCL,
Chlorella powder, or L-carnosine did not show any toxicity;
thus, that dose was selected for those 4 compounds.
e TA-65, vitamin D3, α-lipoic acid, green tea extract, and
rhodiola PE 3%/1% were toxic at doses higher than 50 μg/mL;
therefore, that concentration was used for those compounds.
In addition, the French wine extract showed some
toxicity at a dose of 500 μg/mL, and Astragalus extract
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Ait-Ghezala—Telomerase Activation With Natural Compounds ALTERNATIVE THERAPIES, VOL. 22 NO. S2 9
showed toxicity at a dose of 1000 μg/mL;
therefore, those 2 compounds were tested at
250 μg/mL and 750 μg/mL, respectively.
Glycine, chia seed extract, and broccoli seed
extract did not show any toxicity at the highest
dose tested, 1000 μg/mL, aer 24 hours of
treatment; therefore, those compounds were
subsequently tested at 1000 μg/mL.
e chia seed and Astragalus extract were
poorly soluble and, therefore, the powder was
crushed using a pestle and mortar, and
subsequently resuspended in ethanol and
dimethyl sulfoxide (DMSO), respectively,
before nally being sonicated for 1 minute. e
soluble fraction was collected and then ltered
via a 0.22 μm lter before use. erefore,
although the starting dose of the powder was
50 μg/mL, some of the powder remained
undissolved; thus, the nal dose was an estimate
(data not shown).
Single Compounds
To investigate the telomerase activity in the
single compounds, the research team selected
passages 2, 8 or 10, and 15 to compare the
telomerase activity prole. e data showed that
telomerase activity was reduced with time in the
control cells that were not treated, with P
=
.045
when comparing PD-2 to PD-8 and P
=
.012 when
comparing PD-2 to PD-15 (Figure 1). at nding
was as the research team expected, reecting a
shortening of the telomeres of the primary IMR90
cells because those cells have a nite life span.
e telomerase assay detected a signicant
increase in the telomerase activity for the positive
control, TA-65, with P
=
.0023 and P
=
.007 when
comparing PD-2 to PD-8 and PD-2 to PD-15,
respectively (Figure 1).
Compounds Showing Evidence for
Increasing Telomerase Activity. When
comparing PD-2 to PD-8 for L-carnosine,
N-acetyl-L-cysteine, and DMAE L-bitartrate, no
signicant dierences were detected. However,
when comparing PD-2 to PD-15, those
2 compounds, a signicant increase in telomerase
activity was observed, with P
=
.005 and P
=
.007,
respectively (Figure 1). No trend existed for
DMAE L-bitartrate to increase telomerase
activity when comparing PD-2 to PD-15, and
that measure did not reach statistical signicance,
with P
=
.089 (Figure 1).
Tre at me nt w it h vi ta mi n D 3 showed
signicantly increased telomerase activity when
comparing PD-2 to PD-8, with P
=
.036, and a
marginally signicant increase when comparing
PD-2 to PD-15, with P
=
.047 (Figure 2).
Figure 1. Mean Value of Telomerase Activity With Time at PD-8 and
PD-15 Compared With PD-2, for α-Lipoic Acid, Green Tea Extract,
DMAE L-bitartrate, N-acetyl-L-cysteine HCL, Chlorella Powder, TA-65,
and L-Carnosine
Note: Each error bar was constructed using 1 standard error from the
mean. Statistical signicance was set at α
=
.05 for all statistical analyses.
aFor L-carnosine, P
=
.005 and for N-acetyl-L-cysteine HCL, P
=
.007 for
PD-2 to PD-15, both signicant increases. For TA-65, P
=
.0023 and
P
=
.007, for PD-2 to PD-8 and for PD-2 to PD-15, respectively, both
signicant increases. For α-lipoic acid, P
=
.042 for PD-2 to PD-8 and
P
=
.021 for PD-8 to PD-15, both signicant decreases. For green tea
extract, P
=
.023 for PD-2 to PD-15, a signicant decrease.
Abbreviations: DMAE, dimethylaminoethanol; HCL, hydrochloride.
Figure 2. Mean Value of Telomerase Activity With Time at PD-8 and
PD-15 Compared With PD-2 for Vitamin D3 and Rhodiola PE 3%/1%
Note: Each error bar was constructed using 1 standard error from the
mean. Statistical signicance was set at α
=
.05 for all statistical analyses.
aFor vitamin D3, P
=
.036 for PD-2 to PD-8 and P
=
.047 for PD-2 to PD-15,
both signicant increases. For rhodiola PE 3%/1%, P
=
.066 for PD-2 to
PD-15, an increase indicating a trend toward signicance.
Telomerase Activity % of PD-2
350
300
250
200
150
100
50
0
Control
Green Tea Extract
α-Lipoic Acid
Chlorella Powder
DMAE L-Bitartrate
N-acetyl-L-Carnosine
L-Carnosine
TA-65
a
a
a
a
a
aa
a
a
PD-2
PD-8
PD-15
PD-2
PD-8
PD-15
ControlVitamin D3Rhodiola
PE 3%/1%
a
a
a
250
200
150
100
50
0
Telomerase Activity % of PD-2
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Ait-Ghezala—Telomerase Activation With Natural Compounds
10 ALTERNATIVE THERAPIES, VOL. 22 NO. S2
e rhodiola PE 3%/1% treatment showed a trend toward
an increase in telomerase activity but only for the comparison
of PD-2 to PD-15, with P
=
.066. No signicant change was
detected when comparing PD-2 to PD-8 (Figure 2).
In addition, the results from the telomerase assay
detected a signicant increase in telomerase activity with the
Astragalus extract treatment, with P
=
.0068 when comparing
PD-2 to PD-10 and P
=
.0014 when comparing PD-2 to
PD-15 (Figure 3). When comparing PD-2 to PD-10 for the
broccoli seed extract, no signicant dierences were detected;
however, when comparing P-2 to P-15, a signicant increase
in telomerase activity was observed, with P
=
.018 (Figure 3).
e chia seed extract showed a marginally signicant
increase in telomerase activity, with P
=
.048 when comparing
PD-2 to PD-10, but that eect was lost when comparing
PD-2 to PD-15, with P
=
.07 (Figure 3).
Compounds Showing Evidence for Stabilizing
Telomerase Activity. Chlorella powder did not have any
eect on telomerase activity but also did not show any
evidence of telomere shortening, suggesting that the Chlorella
powder maintained the same level of telomerase activity with
time (Figure 1). A similar eect was seen with the glycine
treatment, suggesting a stabilization of telomerase activity
with time (Figure 3).
Figure 3. Mean Value of Telomerase Activity With Time at
PD-10 and PD-15 Compared With PD-2 for Glycine, French
Wine Extract, Chia Seed Extract, Astragalus Extract, and
Broccoli Seed Extract
Note: Each error bar was constructed using 1 standard error
from the mean. Statistical signicance was set at α
=
.05 for all
statistical analyses.
aFor Astragalus, P
=
.0068 for PD-2 to PD-10 and P
=
.0014 for
PD-2 to PD-15, both signicant increases. For broccoli seed
extract, P
=
.018 for PD-2 to PD-15, a signicant increase.
For chia seed extract, P
=
.048 for PD-2 to PD-10, a
marginally signicant increase, but the eect was lost when
comparing PD-2 with PD-15, with P
=
.07. For French wine
extract, P = .021 for PD-2 to PD-10 and P
=
.003 for PD-2 to
PD-15, both signicant decreases.
Telomerase Activity % of PD-2
200
180
160
140
120
100
80
60
40
20
0
Glycine
Chia Seeds
French Wine
Astragalus
Broccoli Seeds
Control
a
PD-2
PD-8
PD-15
a
aa
a
a
a
Figure 4. Eect of Blends BL-1 to BL-8 on Telomerase
Activity: Mean Value With Time at PD-8 and PD-15
Compared With PD-2
Note: Each error bar was constructed using 1 standard
deviation from the mean. Statistical signicance was set at
α
=
.05 for all statistical analyses.
aFor BL-1, P
=
.022 for PD-2 to PD-8 and P
=
.044 for PD-2 to
PD-15, both signicant increases. For BL-2, P
=
.019 for
PD-2 to PD-8 and P
=
.002 for PD-2 to PD-15, both
signicant increases. For BL-3, P
=
.041 for PD-2 to PD-8
and P
=
.011 for PD-2 to PD-15, both signicant increases.
For BL-4, P
=
.034 for PD-2 to PD-8 and P
=
.011 for PD-2 to
PD-15, both signicant increases. For BL-8, P
=
.028, a
signicant increase for PD-2 to PD-8 only. For BL-5,
P
=
.016 for PD-2 to PD-15, a signicant increase. For BL-6,
P
=
.016 for PD-2 to PD-15, a signicant increase. For BL-7,
P
=
.013 for PD-2 to PD-15, a signicant increase.
Compounds Showing Evidence for Decreasing
Telomerase Activity. Cells treated with α-lipoic acid showed
a similar prole to the control sample, denoting reduced
telomerase activity and, therefore, shorter telomeres. A
signicant decrease in telomerase activity appeared for that
compound when comparing PD-2 to PD-8 and PD-2 to
PD-15, with P
=
.042 and P
=
.021, respectively (Figure 1).
Green tea treatment did not show any eect on
telomerase activity from PD-2 to PD-8, but from PD-2 and
PD-15, a clear reduction in telomerase activity occurred,
with P
=
.023 (Figure 1). Similarly, treatment with French
wine extract revealed a clear decrease in telomerase activity
when comparing PD-2 to PD-10 and PD-2 to PD-15, with
P
=
.021 and P
=
.003, respectively (Figure 3).
Blends of Compounds
Aer the initial screening with the individual compounds,
the research team tested 8 proprietary blends (BL-1 through
BL-8) from the best performing compounds. e results from
the telomerase assay detected a signicant increase in
telomerase activity with the BL-1 blend when comparing PD-2
Telomerase Activity % of PD-2
450
400
350
300
250
200
150
100
50
0
Control
PD-2
PD-8
PD-15
BL-1
BL-2
BL-3
BL-4
BL-5
BL-6
BL-7
BL-8
a
a
a
a
a
a
a
a
a
a
a
a
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Ait-Ghezala—Telomerase Activation With Natural Compounds ALTERNATIVE THERAPIES, VOL. 22 NO. S2 11
to PD-8 and PD-2 to PD-15, with P
=
.022 and P
=
.044,
respectively (Figure 4). A similar trend was found for blends
BL-2, BL-3, and BL-4 when comparing PD-2 to PD-8 and
PD-15, with P
=
.019 and P
=
.002, respectively, for BL-2;
P
=
.041 and P
=
.011, respectively, for BL-3; and P
=
.034 and
P
=
.011, respectively, for BL-4. (Figure 4).
Upon examining BL-8, statistical signicance was
reached only when comparing PD-2 to PD-8, with P
=
.028.
No statistical signicance was observed when comparing
PD-2 to PD-15 (Figure 4). In addition, upon comparing
PD-2 to PD-8 for the BL-5 and BL-6 blends, a trend toward
an increase in telomerase activity was observed. Finally,
statistically signicant dierences were observed for all
3 blends—BL-5, BL-6, and BL-7—when comparing PD-2 to
PD-15, with P
=
.016, P
=
.016, and P
=
.013, respectively
(Figure 4). e data also show that telomerase activity was
reduced with time in the control cells, which were not
treated, when comparing PD-2 to PD-15, with P
=
.08, but the
measure did not reach statistical signicance (Figure 4).
Overall, the data suggested that some blends—BL-1,
BL-2, BL-3, and BL-4—outperformed the other blends. e
blends BL-5, BL-7, and BL-8 were the least ecient at
increasing telomerase activity. BL-6 showed a similar
telomerase activity to the BL-5, BL-7, and BL-8 (Figure 4).
BL-1, BL-2, BL-3, and BL-4 were compared to treatment
with omega-3 FAs, which had previously been shown to
increase telomere length, with decreasing n-6:n-3 omega-3
polyunsaturated fatty acid (PUFA) ratios in a human, double-
blind, 4-month, randomized, controlled trial.26 Because the
current study’s data indicated signicant eects for those rst
4 blends by PD-5, the comparison to omega-3 FAs was
stopped at PD-6 for evaluation of telomerase activity against
PD-1. Overall, the results indicated that the blends
outperformed all of the DHA/EPA doses tested.
e current study’s data showed that the telomerase
activity was reduced with time in the control cells when
comparing P-1 with P-6, with P
=
.044 (Figure 5).
A comparison of the eects of various EPA/DHA doses also
showed that statistical signicance was reached only when
comparing P1 with P6 for the omega-3 dose combination of
EPA 2.5M/DHA 0.25M, with P
=
.024 (Figure 5). No statistical
signicance was observed when comparing other passages,
nor were any dierences observed with the other dose
combinations omega-3 FAs that were tested.
However, a signicant increase in telomerase activity was
detected with BL-2 when comparing PD-1 to PD-4 and PD-1 to
PD-6, with P
=
.032 and P
=
.047, respectively (Figure 5). A
similar trend was found for BL-3 when comparing P-1 to P-2
and P-2 to P-6, with P
=
.024 and P
=
.021, respectively, and for
blend BL-4, when comparing P-2 to P-6 with P
=
.023 (Figure 5).
A trend toward an increase in telomerase activity was
observed when comparing PD-1 and the subsequent passages
of the BL-1 blend (Figure 5); however, no statistical
signicance was reached.
Figure 5. Mean Value of Telomerase Activity With Time at PD-2, PD-8, and PD-15 Compared With PD- 1, for the Best
4 Blends Compared With EPA/DHA
Note: Each error bar was constructed using 1 standard deviation from the mean. Statistical signicance was set at
α
=
.05 for all statistical analyses.
aFor BL-2, P
=
.032 for PD-1 to PD-6 and P
=
.047 for PD-4 to PD-6, both signicant increases. For BL-3, P
=
.024 for PD-1 to
PD-2 and P
=
.021 for PD-2 to PD-6, both signicant increases. For BL-4, P
=
.023 for PD-2 to PD-6, a signicant increase.
Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
Telomerase Activity % of PD-1
250
200
150
100
50
0
Control
BL-1
BL-2
BL-3
BL-4
EPA 25 μM/DHA 2.5 μM
PD-1
PD-2
PD-3
PD-4
EPA 2.5 μM/DHA 0.25 μM
EPA 0.25 μM/DHA 0.025 μM
a
a
a
a
a
a
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Ait-Ghezala—Telomerase Activation With Natural Compounds
12 ALTERNATIVE THERAPIES, VOL. 22 NO. S2
Eects of the 4 Most Potent Blends on Tumor Cell
Proliferation and Survival. No eects occurred for BL-1,
BL-2, BL-3, or BL-4 on viability or proliferation of the A549
cells following 48 hours of treatment (data not shown). In
addition, the potential eects of BL-1, BL-2, BL-3, and BL-4
on the cytotoxicity and the antiproliferative activity of the
chemotherapeutic agent topotecan were further investigated.
e research team found that BL-1, BL-2, BL-3, and BL-4
(Figures 6 and 7) did not aect topotecan’s anticancerous
activity in A549 cells. us, the data indicated that BL-1,
BL-2, BL-3, and BL-4 did not appear to have any eects on
cell toxicity and/or cell proliferation in vitro in A549 cells.
DISCUSSION
Substantial experimental evidence has suggested that cell
senescence is dependent on cell division numbers and that the
total cellular life span is measured by the number of cell
generations, not by chronological time.27,28 Telomere erosion is
prevented by the activation of telomerase, a vital enzyme for
tissue regeneration. It has been hypothesized that telomerase is
suppressed during somatic development; hence, the telomere
length shortens with proliferation.3 Further, investigation of fetal
tissues has shown distinct patterns of regulation, with telomerase
activity remaining longer in the liver, lung, and spleen.29
An aging world population has fueled interest in
regenerative remedies that may address declining organ
function and provide solutions to maintain tness. Reducing
or slowing cellular degeneration, which is in part caused by
telomere erosion, is a key factor in addressing aging at the
cellular level. us, the current research team explored the
potential for natural compounds to slow or reverse the process.
Most of the compounds have previously been described
to have some eects on modulating telomerase activity. For
instance, broccoli seeds,30,31 Chlorella vulgaris extracts,32-34
L-carnosine,35 and vitamin D3
36,37 are all known to aect
telomerase activity. In addition, N-acetyl-L-cysteine,38,39
α-lipoic acids,40-42 green tea extract,43-45 chia seeds,46,47 and
French wine extract are known to aect cellular aging, both
by playing a role in free-radical scavenging and by modulating
telomerase. DMAE, known to have antioxidative properties,
is used as an antiaging agent,48,49 whereas rhodiola is used in
traditional Chinese medicine.50-54
Using a PCR-based assay of telomerase activity, the
current study has shown that N-acetyl-L-cysteine, L-carnosine,
rhodiola PE, vitamin D3, chia seeds, broccoli seeds, and
Astragalus extract all increased telomerase activity when
comparing PD-2 with PD-15 and when comparing those
compounds with the control cells, for which the current study
observed the expected eect of reduced telomerase activity
with time. In addition, DMAE L-bitartrate, Chlorella powder,
and glycine neither increased nor decreased the telomerase
activity, suggesting that they were able to preserve the
telomerase activity from PD-2 to PD-15. However, treatment
of cells with α-lipoic acid, green tea extract, and French red
wine extract appeared to have no benecial eect on telomerase
activity, showing a decrease in telomerase activity with time
that was similar to that observed in the control cells.
Aer investigating the telomerase-modulating eects of
all of the single compounds individually, the current research
created 8 proprietary blends, with dierent concentrations
and compositions of the compounds based on their
performance characteristics during the initial evaluation of
telomerase activity. Although it would be feasible simply to
pick the best individual compound, other factors, such as
synergistic eects, cancer proliferation eects, other adverse
events, reliance on a single material source, and future cost
considerations, made it clear that it was important to study
the combined eects of the compounds.
e current study’s data showed that all blends were
capable of increasing telomerase activity, when comparing
PD-2 to PD-15, with dierent levels of activation, ranging
from 51% to 290% relative to the controls.
Figure 7. Eects of BL-1, BL-2, BL-3, and BL-4 on the
Antiproliferative Activity of the Chemotherapeutic Agent
Topotecan, a Topoisomerase I Inhibitor, on A549 cells, With
48 Hours of Treatment
Figure 6. Eects of BL-1, BL-2, BL-3, and BL-4 on the
Cytotoxicity of Topotecan in A549 Cells, With 48 Hours of
Treatment
Note: BL-1, BL-2, BL-3 and BL-4 did not aect topotecan’s
anticancerous activity or toxicity.
Cytotoxicity (% of control)
200
180
160
140
120
100
80
60
40
20
0
Topotecan Dose (μM)
0 0.1 0.5 1 10
Control
BL-1
BL-2
BL-3
BL-4
Proliferation (% of control)
120
100
80
60
40
20
0
Topotecan Dose (μM)
0 0.1 0.5 1 10
Control
BL-1
BL-2
BL-3
BL-4
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Ait-Ghezala—Telomerase Activation With Natural Compounds ALTERNATIVE THERAPIES, VOL. 22 NO. S2 13
e 4 most potent blends—BL-1, BL-2, BL-3, and
BL-4—all performed better than TA-65, the positive control,
which has been shown to elongate short telomeres and
increase the life span of adult and old mice, without any
increase in cancer incidence.15-20,55 ose 4 blends were
chosen for further investigation with regard to safety (ie, any
eects on cancer proliferation and comparison against
human data on other agents promoting telomerase activity).
In a human, double-blind, 4-month, randomized,
controlled trial,24 treatment with PUFA was shown to increase
telomere length in relation to the decrease in the n-6:n-3
PUFA plasma ratios. Human studies to investigate telomerase
activity and telomere lengthening in humans need to consider
parameters such as sample size and the study’s duration,
which are not well established at the current time because
few such studies have been conducted.
Kiecolt-Glaser et al24 thus have provided important
guidance for the design of future studies because the study
demonstrated changes in telomerase activity and telomere
lengthening its population of 106 individuals in a 4-month
timeframe. In the current study’s in vitro assay, the research
team compared each of 4 blends against the eects of PUFA
combinations. All 4 of the blends outperformed the FA
treatments, which supports their further investigation in
human studies.
Concerns have been voiced as to the potential cancer-
promoting eects of telomerase activators, although to the
current research team’s knowledge, no experimental evidence
exists of such compounds increasing the occurrence of
cancer. Although telomerase does not drive the oncogenic
process, it is permissive and required for the sustained
growth of most advanced cancers. e current study’s tumor
cell proliferation assay provided no evidence for cancer-
promoting eects for its top 4 blends, nor did the blends
interfere with the anticancerous effects of the
chemotherapeutic topotecan.
Although this study has generated important ndings in
the eld of healthy ageing, we are aware of its limitations and
shortcomings. e major limitations herein are listed as
follows: (1) the use of a single cell type, (2) the lack of testing
the eect of the blend of interest beyond PD-15, and (3) the
lack of a companion human study to validate the ndings of
our in vitro experiments. We believe that these limitations
will be addressed and overcome in subsequent work as the
Telomerase Plus blend used in this manuscript is currently
sold as a nutraceutical and can be used in clinical studies to
expand on the existing knowledge of means by which the
ingredients exert their benecial eects.
CONCLUSIONS
e current study’s results have conrmed that many
naturally occurring compounds hold the potential to activate
telomerase and that certain of those compounds have
demonstrated synergistic eects to produce more potent
blends. Given the relationship between telomere shortening,
aging, and the decline of tissue function, it is reasonable to
hypothesize that such telomerase-activating blends may have
health-promoting benets, particularly in relation to aging-
associated conditions. Further investigation of such blends in
human studies that are designed to evaluate safety and the
eects on telomere length are thus warranted.
AUTHOR DISCLOSURE STATEMENT
Funding for the current study was provided by Enzymedica. e Roskamp Institute is
a not-for-prot, public charity, and none of its sta, who comprise all of the authors of
the current paper, received remuneration from Enzymedica. Enzymedica had no role
in the study’s design, data collection or analysis, decision to publish, or preparation of
the manuscript. e authors have declared that no competing interests exist.
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