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Aerobic glycolysis is an established hallmark of cancer. Neoplastic cells display increased glucose consumption and a corresponding increase in lactate production compared to the normal cells. Aerobic glycolysis is regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling pathway, as well as by oncogenic transcription factors such as c-Myc and hypoxia inducible factor 1α (HIF-1α). γ-Tocotrienol is a natural isoform within the vitamin E family of compounds that displays potent antiproliferative and apoptotic activity against a wide range of cancer cell types at treatment doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in mouse +SA and human MCF-7 breast cancer cells. Treatment with γ-tocotrienol resulted in a dose-responsive inhibition of both +SA and MCF-7 mammary tumor cell growth, and induced a relatively large reduction in glucose utilization, intracellular ATP production and extracellular lactate excretion. These effects were also associated with a large decrease in enzyme expression levels involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and lactate dehydrogenase A. γ-Tocotrienol treatment was also associated with a corresponding reduction in the levels of phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but not HIF-1α or glucose transporter 1 (GLUT-1). In summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol are mediated, at least in the part, by the concurrent inhibition of Akt/mTOR signaling, c-Myc expression and aerobic glycolysis.
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1352 Vol. 38, No. 9Biol. Pharm. Bull. 38, 1352–1360 (2015)
© 2015 The Pharmaceutical Society of Japan
Regular Article
Anticancer Effects of γ-Tocotrienol Are Associated with a Suppression in
Aerobic Glycolysis
Parash Parajuli, Roshan Vijay Tiwari, and Paul William Sylvester*
School of Pharmacy, Universit y of Louisiana at Monroe; 700 University Avenue, Monroe, LA 71209, U.S.A.
Received April 3, 2015; accepted May 22, 2015
Aerobic glycolysis is an established hallmark of cancer. Neoplastic cells display increased glucose
consumption and a corresponding increase in lactate production compared to the normal cells. Aerobic
glycolysis is regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin
(mTOR) signaling pathway, as well as by oncogenic transcription factors such as c-Myc and hypoxia induc-
ible factor 1α (H IF-1α). γ-Tocotrienol is a natural isoform within the vitamin E family of compounds that
displays potent antiproliferative and apoptotic activity against a wide range of cancer cel l types at treatment
doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects
of γ-tocotrienol on aerobic glycolysis in mouse SA and human MCF-7 breast cancer cells. Treatment with
γ-tocotrienol resulted in a dose-responsive inhibition of both SA and MCF-7 mammary tumor cell growth,
and induced a relatively large reduction in glucose utilization, intracellular ATP production and extracel-
lular lactate excretion. These effects were also associated with a large decrease in enzyme expression levels
involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate k inase M2,
and lactate dehydrogenase A. γ-Tocotrienol treatment was also associated with a corresponding reduction
in the levels of phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but not HIF-1α or
glucose transporter 1 (GLUT-1). In summary, these findings demonstrate that the antiproliferative effects of
γ-tocotrienol are mediated, at least in the part, by the concurrent inhibition of Akt/mTOR signaling, c-Myc
expression and aerobic glycolysis.
Key words γ-tocotrienol; aerobic glycolysis; breast cancer; vitamin E; c-Myc
Normal differentiated nonproliferating cells metabolize glu-
cose for fuel using glycolysis and oxidative phosphorylation.
However, during hypoxic conditions glycolysis and oxidative
phosphorylation becomes uncoupled and pyruvate is converted
to lactate by anaerobic glycolysis.1,2) In contrast, cancer cells
often metabolize glucose to lactate independently of oxygen
availability using a process call aerobic glycolysis or the
“Warburg effect,” and this characteristic is considered to be
a hallmark of neoplastic transformation.2–5) Although, aerobic
glycolysis is less efficient than oxidative phosphorylation and
requires the conversion of large quantities of glucose to lactate
for the production of ATP, it provides intermediate precursors
for the biosynthesis of macromolecules that are essential for
cancer cell proliferation.1,2)
Various growth factors have been shown to regulate meta-
bolic pathways in both nor mal and cancerous cells.1) Studies
have shown that activation of the phosphatidylinositol-3-kinase
(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway
promotes aerobic glycolysis and increases the expression
of genes responsible for de novo fat ty acid synthesis.1) The
downstream target for Akt, mTOR plays an important role in
the regulation of cell metabolism in response to changes in
nutrient availability and acts to integrates metabolic signals
and cell proliferation by modulating the activation the hypoxia
inducible factor-1α (HIF-1α) and c-Myc,1,6) transcription fac-
tors that are also characteristically elevated in many types of
cancers.7,8)
During conditions of adequate oxygen availability HIF-1α
is degraded by ubiquitination.6, 8) However, during hypoxic
conditions HIF-1α is stabilized and acts to promote aerobic
glycolysis by inducing expression of glucose transporters and
key glycolytic enzymes. HIF-1α also enhances the expression
of lactate dehydrogenase A (LDHA), which acts to prevent py-
ruvate entry into the tricarboxylic acid cycle (TCA) and pro-
mote the production of lactate.1) In contrast, c-Myc regulates
glucose metabolism by increasing the expression of the same
glycolytic enzymes as HIF-1α, but does so during normal,
non-hypoxic conditions.7) Specifically, c-Myc promotes LDHA
activity and contributes to aerobic glycolysis by positively
regulating glucose transporter 1 (GLUT-1), hexokinase II
(HK-II) and phosphofructokinase (PFK).7,9) However, it is now
well established that c-Myc and HIF-1α act in collaboration
to promote aerobic glycolysis in many types of cancer cells.10)
γ-Tocotrienol is a member of the vitamin E family of com-
pounds that displays potent anticancer effects at treatment
doses that have little or no effect on normal cell viability.11 –16 )
Previous studies have shown that γ-tocotrienol significantly
decreases growth factor receptor mediated activation of the
PI 3K /A k t / mT OR p a t hway.14 ,17,18) Recently, it has also been
shown that exposure to tocotrienols decreases c-Myc protein
levels,18 –2 0) and block compensatory increases in HIF-1α dur-
ing hypoxic conditions in cancer cells.21, 22) In addition, com-
bined treatment of γ-tocotrienol with PPARγ antagonists was
found to suppression of the production of adipogenic factors
that are involved in the metabolic reprogramming of mam-
mary cancer cells.23) Taken together, these findings suggests
that γ-tocotrienol may have an effect in modulating aerobic
glycolysis in mammary cancer cells. Therefore, studies were
conducted to determine the effects of γ-tocotrienol on glucose
metabolism and the molecular mechanisms involved in medi-
ating these effects on aerobic glycolysis in the highly malig-
nant mouse +SA mammary tumor and human MCF-7 breast
* To whom correspondence should be addressed. e-mail: sylvester@ulm.edu
Vol. 38, No. 9 (2015) 1353
Biol. Pharm. Bull.
cancer cell lines.
MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich (St. Louis,
MO, U.S.A.), unless otherwise stated. Purified γ-tocotrienol
(>98% purity) was generously provided by First Tech In-
ternational Ltd. (Hong Kong). Antibodies for PI3K, Akt,
phospho-Akt (p-Akt), phospho-mTOR (p-mTOR), HK-II,
LDHA, PFK, and pyruvate kinase muscle isozyme 2 (PKM2)
were purchased from Cell Signaling Technology (Beverly,
MA, U.S.A.). Antibodies specific for c-Myc (9E10), HIF-1α
and monocarboxylate transporter-1 (MCT-1) were purchased
from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.).
Antibodies for GLUT-1, and α-tubulin were purchased from
GeneTex, Inc. (Irvine, CA, U.S.A.). Goat anti-rabbit and goat
anti-mouse secondary antibodies were purchased from Per-
kinElmer, Inc., Biosciences (Boston, MA, U.S.A.).
Mouse +SA mammar y tumor cells are highly malignant,
estrogen independent, and display anchorage-independent
growth when cultured in soft agarose gels.24 –26) B r iefl y, +SA
cells were maintained in serum-free defined control media
consisting of Dulbecco’s modified Eagle’s medium (DMEM/
F12) supplemented with 5 mg/mL bovine serum albumin
(BSA), 10 µg/mL transferrin, 100 U/mL soybean trypsin in-
hibitor, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 ng/
mL EGF, and 10 µg/mL insulin. The estrogen-receptor positive
human MCF-7 breast carcinoma cell line was purchased from
American Type Culture Collection (ATC C, Manassas, VA,
U.S.A.). MCF-7 breast cancer cells were cultured in modi-
fied DMEM/F12 supplemented with 10% fetal bovine serum,
100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 µg/mL
insulin. All cells were incubated at 37°C in an environment
of 95% air and 5% CO2 in humidified incubator. For subcul-
turing, cells were rinsed twice with sterile Ca2+- and Mg2+-
free phosphate-buffered saline (PBS) and incubated in 0.05%
trypsin containing 0.025% ethylenediaminetetraacetic acid
(EDTA) in PBS for 5 min at 37°C. The released cells were
centrifuged, resuspended in serum containing media, and
counted using a hemocytometer.
The stock solution of γ-tocotrienol was prepared by dis-
solving a known amount of γ-tocotrienol in 100 µL of absolute
ethanol and then suspending this solution in sterile 10% BSA
solution followed by gentle shaking at 37°C overnight as de-
scribed previously.11) This stock solution was used to prepare
various concentrations of the treatment media. Ethanol was
added to all treatment groups in a given experiment such
that the nal concentration of ethanol was always maintained
below 0.1%.
For growth studies, (+SA and MCF-7) cells were initially
plated at a density of 5×103 cells/well in 96-well culture plates
(6 wells/group) and returned to incubator and allowed to at-
tach overnight. The next day, cells were divided into differ-
ent treatment groups and then given fresh media containing
various doses of γ-tocotrienol. Fresh media was supplied to
cells in every alternate day during 96 h treatment period.
After wards, viable cell number was determined using the
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay, as described previously.21,27 ) Brief-
ly, media was replaced in all treatment groups with fresh con-
trol media containing 0.5 mg⁄mL MTT. After a 3 h incubation
period, medium was removed, MTT crystals were dissolved
in dimethyl sulfoxide (DMSO) (100 µL/well), and optical den-
sity of each sample was measured at 570 nm on a microplate
reader (SpectraCount, Packard BioScience Company, Meriden,
CT, U.S.A.) zeroed against a blank prepared from cell-free
medium. Numbers of cells/well were calculated against a
standard curve prepared by plating known cell densities, as
determined by hemocytometer, in triplicate at the beginning
of each experiment.
For the measurement of intracellular ATP levels, (+SA and
MCF-7) cells were initially plated at a density of 5×103 cells/
well in 96-well culture plates (6 wells/group) and allowed to
attach overnight. The next day, cells were divided into differ-
ent treatment groups and then given fresh media containing
various doses of γ-tocotrienol. Cells in all groups were fed
fresh treatment media every other day for a 96 h treatment
period. Afterwards, intracellular ATP levels were determined
using the CellTitre-Glo luminescence assay Promega Corpora-
tion (Madison, WI, U.S.A.) according to the manufacturer’s
instructions.
For the measurement of glucose consumption from cul-
ture media, (+SA and MCF-7) cells were plated at a density
of 1×105 cells in 6-well culture plate and allowed to attach
overnight. The following day, cells were divided into dif-
ferent treatment groups (3 wells/group), culture media was
removed, washed with sterile PBS, cells were treated with
0–8 µM γ-tocotrienol and fresh media was fed every other day
during the 4-d culture period and glucose concentration was
measured as described previously.28) Briefly, culture media
was removed from the culture plates and glucose concentra-
tions (mg/dL) were measured using an ACCU-CHEK Active
glucose meter (Roche Diagnostics, Indianapolis, IN, U.S.A.).
Glucose concentrations were calculated by subtracting the glu-
cose concentration in each treatment groups from the glucose
concentration from the cell-free media blank.
For the measurement of extracellular lactate excreted into
culture media, cells were plated at a density of 1×105 cells
in 6-well culture plate and allowed to attach overnight. The
following day, cells were divided into different treatment
groups (3 wells/group), culture media was removed, washed
with sterile PBS, cells were treated with 0 –8 µM γ-tocot rienol
and fresh media was fed every other day during the 4-d cul-
ture period. Afterwards, lactate was measured using L-lactate
Assay Kit (Cayman Chemical Co., Ann Arbor, MI, U.S.A.).
Briefly, 500 µL media was collected from each control and
treatment groups and the supernatants were neutralized with
25 µL of 5
M K2CO3 provided in the kit, then centrif uged for
5 min at 10000×g, and then diluted 1 : 2 v/v with assay buffer.
Standard solutions and sample aliquots were transferred to 96-
well plates, and mixed with 100 µL assay buffer, 20 µL cofac-
tor mixture, and 20 µL fluorometric substrate. Reactions were
then initiated by the addition of 40 µL enzyme mixture and
then allowed to incubate for 20 min. Fluorescence was mea-
sured using excitation and emission wavelengths of 530 nm
and 590 nm, respectively.
For Western blot analysis, +SA and MCF-7 cells, were plat-
ed at the density of 1×106 cells, allowed to attach overnight.
The following day, cells were washed with PBS and exposed
to control or treatment media containing 0–8 µM γ-tocotrienol
for a 4-d culture period. Afterwards, the cells were isolated
with trypsin, washed in PBS, then whole cell lysates were pre-
1354 Vol. 38, No. 9 (2015)
Biol. Pharm. Bull.
pared as described previously, in Laemmli buffer.29,3 0) Protein
concentration in each sample was determined using Bio-Rad
protein assay kit (Bio Rad, Hercules, CA, U.S.A.). Equal
amounts of protein (30–50 µg/lane) from each sample were
then subjected to electrophoresis through 7.5–20% sodium
dodecyl sulfate (SDS)-polyacrylamide minigels. Proteins from
minigels were transplotted at 30 V for 12–16 h at 4°C onto
a single 8×6.5 polyvinylidene fluoride (PVDF) membrane
(PerkinElmer, Inc., Life Sciences, Wellesley, MA, U.S.A.)
in a Trans-Blot Cell (Bio-Rad) according to the methods of
Tow b i n . 31 ) These PVDF membranes were then blocked with
2% BSA in 10 mM Tris–HCl containing 50 mM NaCl and 0.1%
Tween 20, pH 7.4 (TBST) then incubated with specific pri-
mary antibodies against PI3K, Akt, p-Akt, p-mTOR, c-Myc,
HI F-1α, HK-II, PFK, PKM2, LDHA, GLUT-1, and MCT-1
diluted 1 : 1000 to 1 : 5000 in 2% BSA in TBST overnight at
4°C. Membranes were incubated in primar y antibodies against
α-tubulin, diluted 1 : 5000 in 2% BSA in TBST for 2 h at
room temperature. Membranes were then washed 5 times in
TBST and incubated with respective horseradish peroxide-
conjugated secondary antibody diluted 1 : 5000 in 2% BSA
in TBST for 1 h at room temperature followed by washing in
TBST 5 times. Blots were then visualized by chemilumines-
cence according to the manufacturer’s instructions (Pierce,
Rockford, IL, U.S.A.). Images of protein bands from all treat-
ment groups within a given experiment were acquired using
Syngene Imaging System (Frederick, MD, U.S.A.). Visualiza-
tion of α-tubulin was used to ensure equal sample loading in
each lane. All experiments were repeated at least 3 times and
a representative Western blot image from each experiment are
shown in the figures.
Differences between groups were analyzed by one-way
ANOVA followed by Dunnett’s t-test. A difference of p<0.05
was considered statistically significant as compared to the ve-
hicle-treated control group or as defined in the figure legends.
RESULTS
Effects of Various Doses of γ-Tocotrienol on Mouse
+SA and Human MCF-7 Mammary Tumor Cell Vi-
ability Treatment with 3– 4 µM (+SA) or 6–8 µM ( MCF-7)
γ-tocotrienol for a 96 h culture period significantly inhibited
mammary cancer cell growth in dose-dependent manner as
compared with the cells in their corresponding vehicle-treated
control group (Fig. 1).
Effects of γ-Tocotrienol on Media Glucose, Intracellular
ATP, and Extracellular Lactate Concentrations in +SA
and MCF-7 Cancer Cells Based on previously conducted
dose– and time–response studies examining the relationship
between effects of γ-tocotrienol on c-Myc expression and the
growth of +SA and MCF-7 mammary tumor cells,18) a 96 h
exposure period was selected for the present st udies. Treat-
ment with 3– 4 µM (+SA) or 6–8 µM ( MCF-7) γ-tocotrienol for
a 96 h culture period significantly reduced mammary cancer
cell glucose consumption in a dose-dependent manner as com-
pared to their corresponding vehicle-treated control groups
(Fig. 2A). This same treatment was also found to significantly
inhibit intracellular ATP levels in a dose-dependent manner in
both +SA and MCF-7 mammary cancer cells as compared to
their corresponding vehicle-treated control groups (Fig. 2B).
Treatment with 4 µM (+SA) or 8 µM (MCF-7) γ-tocotrienol for
a 96 h culture period significantly reduced extracellular lactate
levels as compared to their corresponding vehicle-treated con-
trol groups (Fig. 2C).
Effects of γ-Tocotrienol on Intracellular Levels of HK-II,
PFK, PKM2 and LDHA in +SA and MCF-7 Mammary
Cancer Cells Western blot analysis showed that following
a 96 h culture period, treatment with 2–4 µM (+SA) or 4–8 µM
(M CF-7 ) γ-tocotrienol induced a dose-responsive dependent
decrease in intracellular levels of HK-II, PFK, PKM2 and
LDHA in +SA (Fig. 3A) and MCF-7 (Fig. 3B) as compared to
their respective vehicle-treated control groups. Scanning den-
sitometric analysis of protein bands in each blot showed that
treatment with 4 µM (+SA) and 8 µM ( MC F-7) γ-tocot rienol
induced a significant decrease in total HK-II, PFK, PKM2 and
LDHA levels as compared to cells in their respective vehicle-
treated control groups (Figs. 3A, B). Western blot analysis also
showed that similar treatment with 2– 4 µM (+SA) or 4–8 µM
Fig. 1. Antiproliferative Effects of γ-Tocot rienol on +SA and MCF-7 Mamma ry Tumor Cells
Vertical ba rs ind icate mea n cell number±S.E.M. i n each tr eatme nt group. * p<0.05 as c ompare d to cells i n their r espective vehicle-treated cont rol group.
Vol. 38, No. 9 (2015) 1355
Biol. Pharm. Bull.
(M CF-7 ) γ-tocotrienol had no effect on GLUT-1, but induced
a dose-dependent decrease in MCT-1 levels in +SA (Fig. 3C)
and MCF-7 (Fig. 3D) mammary cancer cells as compared to
their respective vehicle-treated control groups. Scanning den-
sitometric analysis of protein bands in each blot showed that
treatment with 4 µM (+SA) and 8 µM ( MC F-7) γ-tocot rienol
induced a significant decrease in GLUT-1 and MCT-1 levels
as compared to cells in their respective vehicle-treated control
groups (Figs. 3C, D).
Effects of γ-Tocotrienol on PI3K/Akt/mTOR Signaling,
and c-Myc and HIF-1α Transcription Factor Levels in +SA
and MCF-7 Mammary Cancer Cells Western blot analysis
showed that treatment with 2–4 µM (+SA) or 4–8 µM (M CF-7)
of γ-tocotrienol induced a dose-dependent decrease in PI3K,
phospho-Akt (active), phospho-mTOR (active) and c-Myc lev-
els in +SA (Fig. 4A) and MCF-7 (Fig. 4B) mammary cancer
cells as compared to their respective vehicle-treated control
groups. However, HIF-1α protein expression was found to be
nearly undetectable, and exposure to γ-tocotrienol had little or
no effect on HIF-1α expression in either +SA or MCF-7 mam-
mary cancer cells (Figs. 4A, B).
DISCUSSION
Results in the present study demonstrate that the antipro-
liferative effects of γ-tocotrienol are associated with a suppres-
sion in aerobic glycolysis characterized by a significantly large
reduction in glucose consumption, ATP production and corre-
Fig. 2. Effects of γ-Tocotrienol on Media Glucose, Intracellular ATP and Extracellular Lactate Levels in +SA and MCF-7 Mammary Cancer Cells
(A) Glucose was mea sured in a 200 µL sample obta ined from each well u sing a st andard glucometer. Vertical bars re present m ean gluc ose consumption (mg/dL)±S.E.M.
in each t reatment grou p. (B) Intracellul ar ATP level was de term ined u sing the lu mine scence a ssay. Vertical bars ind icate mea n relative p ercent age of ATP in control
group±S.E.M. in each t reat ment group. (C) Extra cellula r lactate was mea sured using th e L-lacta te assay k it. Vertica l bars i ndicate mean relat ive perce ntage of lac tate i n
control group±S.E.M. in each t reatment group. * p<0.05 as compar ed to cell s in thei r respe ctive vehicle -tre ated cont rol group.
1356 Vol. 38, No. 9 (2015)
Biol. Pharm. Bull.
sponding decrease in lactate excretion in both mouse +SA and
human MCF-7 mammary cancer cells. These γ-tocotrienol-
induced effects were also found to be directly related to a de-
crease in the expression of key glycolytic enzymes including
HK-II, PKM2, PFK and LDHA, a corresponding decrease in
PI3K/Akt/mTOR and c-Myc expression, signaling factors that
play a key role in modulating aerobic glycolysis in these mam-
mary cancer cells.
Previous studies showed that mitochondria targeting
synthetic analogs of α-tocopherol can directly inhibit gly-
colysis and reduce intracellular ATP levels in MCF-7 and
MDA-MB-231 breast cancer cells.32) However, the molecular
mechanism of action mediating the effects of these tocoph-
erol analogs had not been determined. The present study
Fig. 3. Wester n Blot Analysis of γ-Tocot rienol Effects on the Relative Protein Levels of Key Aerobic Glycolytic Proteins a nd Glucose and Lactate
Transpor ters i n +SA (A and C) and MCF-7 (B and D) Mammary Tumor Cells
Afte r exper iment ation, whole cell lysat es were pre pared for subsequ ent sepa ration by p olyacr ylamide gel electr ophoresis (50 µg/ lane) followed by Weste rn blot an alysis
for HK-II, PFK, PKM2, L DHA, GLUT-1 and MCT-1. The v isual izat ion of α-tubu lin was u sed to en sure equal sample loadi ng in each lane. All experiments we re repe ated
at least 3 times. Scanni ng densit ometr ic analy sis was performe d for each blot to visualize th e relative level s of proteins. Integ rate d optical d ensity of e ach band wa s nor-
maliz ed with t heir cor responding α-tubul in and control tr eatme nt bands a nd then shown in bar graph s. Vertical b ars ind icate th e percentage cha nge in prot ein levels i n the
various treat ment groups±S.E.M. as compared with their res pective vehicle-t reated control g roup. * p<0.05 as com pared to their re spect ive vehicle-treat ed control group.
Vol. 38, No. 9 (2015) 1357
Biol. Pharm. Bull.
confirms and extends these previous finding by demonstrating
that γ-tocotrienol, a natural vitamin E isoform, significantly
inhibits mitogenic/metabolic signaling pathways that are in-
volve in regulating glucose metabolism, and ATP and lactate
production by suppressing the activity of specific glycolytic
enzymes in both mouse +SA and human MCF-7 mammary
cancer cells.
It is well established that hexokinase catalyzes ATP-depen-
dent conversion of glucose to glucose-6-phosphate and is the
initial enzymatic step in glycolysis.33) There are 4 different
hexokinase isomers (I–IV) that are expressed in the mam-
malian tissues, but HK-II expression is characteristically very
high in cancer cells.34) Studies in HK-II conditional knockout
mice showed that this enzyme is essential for tumor survival
in Ras mutation-dependent lung cancer and ErbB2 mutation-
dependent breast cancer.34) Furthermore, knockout of HK-II
resulted in significant growth inhibition of these cancer cells
in both in vivo and in vitro experimental models.34) The pres-
ent study shows that the anticancer effects of γ-tocotrienol are
directly related with a corresponding decrease in HK-II levels
and reduction in glucose metabolism.
PFK catalyzes the conversion of fructose-6-phosphate to
fructose 1,6-bisphosphate and is the rate limiting steps of
glycolysis. Akt has also been shown to positively regulate
PF K acti v ity.35) Results show that γ-tocotrienol treatment
induces a relatively large reduction in phosphorylated (acti-
vated) Akt levels and this effect was directly associated with
a corresponding reduction in PFK levels in both +SA and
MCF-7 mammary cancer cells. The final rate limiting step
in glycolysis is the conversion of phosphoenol pyruvate to
pyruvate and is catalyzed by the enzyme pyruvate kinase.2)
There are 4 different isoforms of pyruvate kinase (muscle type
1, muscle type 2, liver; and red blood cell) that are expressed
in mammalian tissues.36) Studies have shown that the cancer
cells selectively overexpress pyruvate kinase muscle type 2,
an isoform that is predominately expressed during embryogen-
esis, whereas nor mal differentiated nonproliferating cells pri-
marily express pyruvate kinase muscle type 1.37) The PKM2
and PKM1 isoforms appear to be mutually exclusive splice
variants from the same gene called PKM, and studies have
shown that the preferential expression of PKM2 in cancer
cells is mediated through a c-Myc-dependent mechanism.38, 39)
Fig. 4. Wester n Blot Analysis of γ-Tocot rienol Effects on the Relative Protein Levels of PI3K, A kt, mTOR, c-Myc and HIF-1α in +SA (A) and
MCF-7 (B) Mammary Tumor Cells
Whole cell lysates were prepa red for subsequent s epara tion by polya cryla mide gel elec trophoresis (50 µg /lane) followed by Western blot a nalysis . The visualiz ation of
α-tubulin was u sed to en sure eq ual sam ple loadi ng in each lane. All experi ments were repea ted at lea st 3 time s. Scan ning de nsitomet ric an alysis was perfor med for ea ch
blot to visu alize the relat ive levels of proteins. Integrated optic al densit y of each ba nd was nor malized with t heir cor resp onding α-tubulin and control t reatment bands and
then show n in bar g raphs. Ver tical ba rs indicate the p ercent age change in protein levels in various t reat ment groups±S.E.M. as co mpared with their respective veh icle-
treat ed control group. * p<0.05 as c ompare d to thei r respective vehicle -treated cont rol grou p.
1358 Vol. 38, No. 9 (2015)
Biol. Pharm. Bull.
Furthermore, PKM2 can induce elevations in c-Myc expres-
sion through a positive feedback mechanism, resulting in a
large increase in LDHA levels and activity, and is responsible
for the amplification of aerobic glycolysis in cancer cells.4 0)
Reports have also identified mTOR as a central activator of
aerobic glycolysis in non-hypoxic conditions by stimulating
c-Myc-mediated PKM2 gene splicing, whereas treatments that
inhibit mTOR activity were found to cause a suppression in
PKM2 expression.41) Recent studies have also shown that the
antiproliferative effects of γ-tocotrienol on +SA and MCF-7
mammary tumor cells is associated with a dose- and time-
dependent reduction in c-Myc protein and mRNA expression,
and Akt/mTOR activity.18) The present study extends these
previous findings and demonstrates that γ-tocotrienol-induced
reductions c-Myc expression and Akt/mTOR activity is associ-
ated with a cor responding decrease in PKM2 protein levels.
Presently, it has not yet been determined if γ-tocotrienol act
directly or indirectly by suppressing Akt/mTOR signaling to
reduce c-Myc expression. Nevertheless, these findings sug-
gest that γ-tocotrienol attenuates c-Myc-dependent preferential
splicing of PKM2 and consequently lead to suppression of
aerobic glycolysis. However, additional experiments are re-
quired to confirm this hypothesis.
In the final step of glycolysis, LDHA catalyzes the conver-
sion of pyruvate to lactate. LDHA expression has also been
shown to be enhanced by oncogenic c-Myc activity.7,42) Previ-
ous studies have demonstrated that cancer cells produce more
lactate and display higher levels of LDHA than normal cells,
and treatments that silence LDHA expression and/or activ-
ity, significantly inhibit of breast cancer cell proliferation.43)
Lactate generated during glycolysis is excreted out of cells by
monocarboxylate transporters and oncogenic c-Myc activity
increases MCT-1 expression in several types of cancers.44, 45)
In addition, treatments that inhibit c-Myc activity induce a
reduction in MCT-1 expression and a subsequent reduction in
cancer cell aerobic glycolysis and proliferation.4 4,45) Results
show that mammary cancer cells exposed to γ-tocotrienol
treatment displayed a significant decrease in c-Myc, LDHA
and MCT-1 levels and a cor responding reduction in lactate
production. These ndings strongly suggest that γ-tocotrienol-
induced inhibition in c-Myc expression plays an impor tant
role in mediating γ-tocotrienol action in suppressing aerobic
glycolysis and cancer cell proliferation. In summary, these
findings suggest that γ-tocotrienol-induced suppression of
aerobic glycolysis in +SA mouse mammar y and MCF-7
human breast cancer cells is associated with suppression of
key regulatory enzymes (HK-II, PFK, PKM2 and LDHA)
and signaling proteins (p-Akt, p-mTOR and c-Myc) involved
in the regulation of glycolysis. The intracellular mechanisms
involved in mediating γ-tocotrienol effects in the suppression
of aerobic glycolysis are summarized in Fig. 5.
Acknowledgments This work was supported in part by
Grants from First Tec International Ltd. (Hong Kong), the
Malaysian Palm Oil Council (MPOC), the Louisiana Cancer
Foundation and the Louisiana Campuses Research Initiative
(L ACRI ).
Conflict of Interest The authors declare that they have no
personal, financial or competing conflict of interest. First Tech
International Ltd. provided a Grant that partially paid for the
funding of these experiments and purified γ-tocotrienol that
Fig. 5. Schematic Representation of γ-Tocotrienol-Induced Suppression of Aerobic Glycolysis
Vol. 38, No. 9 (2015) 1359
Biol. Pharm. Bull.
was used in these experiments.
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... γ-Tocotrienol-induced c-Myc degradation was also found to be associated with GSK-3β activation and proteasomal function. Furthermore, cancer cells display increased glucose consumption and a corresponding increase in lactate production even in the presence of oxygen compared to the normal cells, a hallmark of the Warburg effect or aerobic glycolysis (Parajuli et al., 2015b). Aerobic glycolysis is regulated by the activation of the PI3K/Akt/mTOR pathway and oncogenic transcription factors, such as c-Myc and the HIF-1α. ...
... Aerobic glycolysis is regulated by the activation of the PI3K/Akt/mTOR pathway and oncogenic transcription factors, such as c-Myc and the HIF-1α. Studies conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in human MCF-7 breast cancer cells showed that treatment with 0À10 μM γ-tocotrienol resulted in a dose-responsive inhibition of MCF-7 cell growth (Parajuli et al., 2015b). This treatment was also found to result in a relatively large reduction in glucose utilization and a corresponding decrease in intracellular ATP production and extracellular lactate excretion by MCF-7 mammary tumor cells (Parajuli et al., 2015b). ...
... Studies conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in human MCF-7 breast cancer cells showed that treatment with 0À10 μM γ-tocotrienol resulted in a dose-responsive inhibition of MCF-7 cell growth (Parajuli et al., 2015b). This treatment was also found to result in a relatively large reduction in glucose utilization and a corresponding decrease in intracellular ATP production and extracellular lactate excretion by MCF-7 mammary tumor cells (Parajuli et al., 2015b). Fig. 20.3 shows Western blot analysis of γ-tocotrienol anticancer effects showing that these effects are also associated with a large decrease in the expression of intracellular enzymes involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and LDHA, in MCF-7 human breast cancer cells (Parajuli et al., 2015b). ...
... The anticancer activity of c-tocotrienol in breast cancer cells has previously been shown to involve the modulation of a wide range of signaling mechanisms involved in regulating proliferation, survival and metabolism (14,(16)(17)(18)(19)(20)(21). Studies have also shown that c-tocotrienol treatment is associated with a decrease in glycolytic enzymes levels in breast cancer cells (22). Therefore, studies were conducted in to investigate the effects of c-tocotrienol on the Warburg effect in human breast cancer cells. ...
... All cells were fed treatment media every other day for the 4-day experimental period. A 4-day cell culture incubation period was selected based on numerous studies conduct in our laboratory that determined that a 4-day culture period is optimal for measuring maximum cell proliferation and c-tocotrienol treatment effects in MCF-7 and MDA-MB-231 cell lines (16)(17)(18)(22)(23)(24). For subculturing and use in experimentation, cells were isolated by first being rinsed in sterile Ca þþ and Mg þþ free phosphate-buffered saline (PBS), and then incubated in 0.05% trypsin with 0.025% EDTA in PBS for 5 min at 37 C. Cells were then collected and centrifuged, resuspended in fresh medium and counted manually using a hemocytometer. ...
... The next day, media was removed, cell divided into different treatment groups (three wells/group) and then cells were exposed to their respective treatment media for a 4-day experimental period. Glucose levels (mg/dl) in all wells were measured at the end of experimentation using an ACCU-CHEK Active glucose meter (Roche Diagnostics, Indianapolis, IN) as described previously (22). Glucose concentrations were calculated by subtracting the glucose concentration in each treatment groups from the glucose concentration obtain from media in the cell-free blank wells. ...
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... In agreement with these observations, treatment with Rapamycin (an inhibitor of mTOR) is able to suppress the SASP in various model of senescent cells. In analogy with the effects of Rapamycin, a mixture of naturally occurring T3 and tocopherols extracted from palm oil/ palm fruits as well as purified γ-T3 has been reported to negatively modulate mTOR pathways in breast cancer cells [57,83]. Moreover, T3 have been reported to suppress the activation of NF-kB in several experimental models (see previous chapter focused on T3 and inflammation) [66,79,89]. ...
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