2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma
Aerobic glycolysis that supports high proliferation rate and survival of tumor cells in unfavorable conditions is among fundamental features of tumor metabolism. The search for active modulators of energetic metabolism capable of suppressing tumor growth and metastasis could result in higher effecti...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2016
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irk-123456789-1377172020-11-12T11:03:54Z 2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma Pyaskovskaya, O.N. Kolesnik, D.L. Fedorchuk, A.G. Prochorova, I.V. Solyanik, G.I. Original contributions Aerobic glycolysis that supports high proliferation rate and survival of tumor cells in unfavorable conditions is among fundamental features of tumor metabolism. The search for active modulators of energetic metabolism capable of suppressing tumor growth and metastasis could result in higher effectiveness of anticancer therapy. Aim: To study antitumor and antimetastatic activity of the modulators of energetic metabolism dichloroacetate (DCA) and 2-deoxy-D-glucose (2DG) used in combination treatment of Lewis lung carcinoma (LLC). Materials and Methods: As experimental tumor model, LLC/R9 variant was used. DCA and 2DG were administered per os to С57Bl/6 mice 5 times per week for 3 weeks at a total dose of 1.5 and 0.98 g/kg, respectively, as single agents or in combination starting from the following day after tumor cell transplantation. Growth of primary tumor and number and volume of lung metastases were registered. Lactate and pyruvate content was determined by enzymatic methods using lactate dehydrogenase. Electron paramagnetic resonance was used for analyzing the functional state of the components of mitochondrial respiratory chain. Engulfing activity and reactive oxygen species (ROS) production in tumor-associated СD14⁺cells was analyzed by flow cytometer with the use of FITC-labeled staphylococcus, and by spectrofluorometry with the use of 2.7-dichlorofluorescein diacetate, respectively. Results: DCA administered as a single agent did not affect primary tumor growth but decreased the number and volume of lung metastases by 60% (p < 0.05) and 90% (p < 0.05), respectively. In mice treated with 2DG only, primary tumor volume as well as the number and volume of lung metastases were not affected. Combination treatment with DCA and 2DG resulted in the decrease of primary tumor volume, the number and volumes of lung metastases by 70; 46, and 90%, respectively (р < 0.05). High antitumor activity of DCA + 2DG was associated with 31% decrease (p < 0.05) of lactate content in tumor tissue and 120% increase (p < 0.01) of ROS production in СD14⁺ cells recruited to the region of tumor growth. Conclusion: 2DG that possesses neither antitumor nor antimetastatic activity against LLC/R9 significantly enhanced antitumor activity of DCA with accompanying inhibition of glycolysis and increase of cytotoxic activity of СD14⁺cells infiltrating tumor tissue. Taking into account significant antimetastatic activity of DCA this substance could be considered as a promising antimetastatic agent. 2016 Article 2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma / O.N. Pyaskovskaya, D.L. Kolesnik, A.G. Fedorchuk, I.V. Prochorova, G.I. Solyanik // Experimental Oncology. — 2016 — Т. 38, № 3. — С. 176–180. — Бібліогр.: 27 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137717 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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English |
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Original contributions Original contributions |
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Original contributions Original contributions Pyaskovskaya, O.N. Kolesnik, D.L. Fedorchuk, A.G. Prochorova, I.V. Solyanik, G.I. 2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma Experimental Oncology |
| description |
Aerobic glycolysis that supports high proliferation rate and survival of tumor cells in unfavorable conditions is among fundamental features of tumor metabolism. The search for active modulators of energetic metabolism capable of suppressing tumor growth and metastasis could result in higher effectiveness of anticancer therapy. Aim: To study antitumor and antimetastatic activity of the modulators of energetic metabolism dichloroacetate (DCA) and 2-deoxy-D-glucose (2DG) used in combination treatment of Lewis lung carcinoma (LLC). Materials and Methods: As experimental tumor model, LLC/R9 variant was used. DCA and 2DG were administered per os to С57Bl/6 mice 5 times per week for 3 weeks at a total dose of 1.5 and 0.98 g/kg, respectively, as single agents or in combination starting from the following day after tumor cell transplantation. Growth of primary tumor and number and volume of lung metastases were registered. Lactate and pyruvate content was determined by enzymatic methods using lactate dehydrogenase. Electron paramagnetic resonance was used for analyzing the functional state of the components of mitochondrial respiratory chain. Engulfing activity and reactive oxygen species (ROS) production in tumor-associated СD14⁺cells was analyzed by flow cytometer with the use of FITC-labeled staphylococcus, and by spectrofluorometry with the use of 2.7-dichlorofluorescein diacetate, respectively. Results: DCA administered as a single agent did not affect primary tumor growth but decreased the number and volume of lung metastases by 60% (p < 0.05) and 90% (p < 0.05), respectively. In mice treated with 2DG only, primary tumor volume as well as the number and volume of lung metastases were not affected. Combination treatment with DCA and 2DG resulted in the decrease of primary tumor volume, the number and volumes of lung metastases by 70; 46, and 90%, respectively (р < 0.05). High antitumor activity of DCA + 2DG was associated with 31% decrease (p < 0.05) of lactate content in tumor tissue and 120% increase (p < 0.01) of ROS production in СD14⁺ cells recruited to the region of tumor growth. Conclusion: 2DG that possesses neither antitumor nor antimetastatic activity against LLC/R9 significantly enhanced antitumor activity of DCA with accompanying inhibition of glycolysis and increase of cytotoxic activity of СD14⁺cells infiltrating tumor tissue. Taking into account significant antimetastatic activity of DCA this substance could be considered as a promising antimetastatic agent. |
| format |
Article |
| author |
Pyaskovskaya, O.N. Kolesnik, D.L. Fedorchuk, A.G. Prochorova, I.V. Solyanik, G.I. |
| author_facet |
Pyaskovskaya, O.N. Kolesnik, D.L. Fedorchuk, A.G. Prochorova, I.V. Solyanik, G.I. |
| author_sort |
Pyaskovskaya, O.N. |
| title |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| title_short |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| title_full |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| title_fullStr |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| title_full_unstemmed |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| title_sort |
2-deoxy-d-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2016 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137717 |
| citation_txt |
2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma / O.N. Pyaskovskaya, D.L. Kolesnik, A.G. Fedorchuk, I.V. Prochorova, G.I. Solyanik // Experimental Oncology. — 2016 — Т. 38, № 3. — С. 176–180. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T02:38:08Z |
| last_indexed |
2025-07-10T02:38:08Z |
| _version_ |
1837225833995960320 |
| fulltext |
176 Experimental Oncology 38, 176–180, 2016 (September)
2-DEOXY-D-GLUCOSE ENHANCES DICHLOROACETATE
ANTITUMOR ACTION AGAINST LEWIS LUNG CARCINOMA
O.N. Pyaskovskaya*, D.L. Kolesnik, A.G. Fedorchuk, I.V. Prochorova, G.I. Solyanik
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aerobic glycolysis that supports high proliferation rate and survival of tumor cells in unfavorable conditions is among fundamental
features of tumor metabolism. The search for active modulators of energetic metabolism capable of suppressing tumor growth and
metastasis could result in higher effectiveness of anticancer therapy. Aim: To study antitumor and antimetastatic activity of the
modulators of energetic metabolism dichloroacetate (DCA) and 2-deoxy-D-glucose (2DG) used in combination treatment
of Lewis lung carcinoma (LLC). Materials and Methods: As experimental tumor model, LLC/R9 variant was used. DCA and 2DG
were administered per os to С57Bl/6 mice 5 times per week for 3 weeks at a total dose of 1.5 and 0.98 g/kg, respectively, as single
agents or in combination starting from the following day after tumor cell transplantation. Growth of primary tumor and number
and volume of lung metastases were registered. Lactate and pyruvate content was determined by enzymatic methods using lactate
dehydrogenase. Electron paramagnetic resonance was used for analyzing the functional state of the components of mitochondrial
respiratory chain. Engulfing activity and reactive oxygen species (ROS) production in tumor-associated CD14+ cells was analyzed
by flow cytometer with the use of FITC-labeled staphylococcus, and by spectrofluorometry with the use of 2.7-dichlorofluorescein
diacetate, respectively. Results: DCA administered as a single agent did not affect primary tumor growth but decreased the number
and volume of lung metastases by 60% (p < 0.05) and 90% (p < 0.05), respectively. In mice treated with 2DG only, primary tumor
volume as well as the number and volume of lung metastases were not affected. Combination treatment with DCA and 2DG re-
sulted in the decrease of primary tumor volume, the number and volumes of lung metastases by 70; 46, and 90%, respectively
(р < 0.05). High antitumor activity of DCA + 2DG was associated with 31% decrease (p < 0.05) of lactate content in tumor tissue
and 120% increase (p < 0.01) of ROS production in СD14+ cells recruited to the region of tumor growth. Conclusion: 2DG that
possesses neither antitumor nor antimetastatic activity against LLC/R9 significantly enhanced antitumor activity of DCA with
accompanying inhibition of glycolysis and increase of cytotoxic activity of CD14+ cells infiltrating tumor tissue. Taking into account
significant antimetastatic activity of DCA this substance could be considered as a promising antimetastatic agent.
Key Words: Lewis lung carcinoma, antitumor and antimetastatic action, dichloroacetate, 2-deoxy-D-glucose, reactive oxygen
species, CD14+ macrophage-like cells.
It is well known that glycolysis is the main way
of ATP generation in the large majority of malignant
cells even in the presence of oxygen. Despite the
fact that special features of tumor bioenergetics are
being used in oncological practice for diagnostic and
prognostic purposes since 50th of last century until
now [1], only recently tumor metabolism has started
to be considered as a therapeutic target [2]. As soon
as glycolysis has been recognized as the main meta-
bolic way providing tumor cell proliferation and survival
in unfavorable conditions, the research has been fo-
cused on the compounds capable to suppress tumor
growth via modification of its energetic metabolism.
Among such compounds one could mention sodium
dichloroacetate (DCA), an inhibitor of pyruvate de-
hydrogenase (PDH) kinase, and 2-deoxy-D-glucose
(2DG), an inhibitor of glycolysis. Usually DCA has been
used for correction of chronic metabolic malfunctions,
but several data [3, 4] prompted to consider DCA
as a promising anticancer agent capable of activating
indirectly the enzymes of PDH complex and conse-
quently shifting tumor cell metabolism from glycolysis
to oxidative phosphorylation. 2DG is a structural ana-
log of glucose in which hydroxyl group in the second
position is replaced with hydrogen. Due to its structure,
2DG is irreversibly phosphorylated with hexokinase
into 2DG-phosphate that is accumulated in a cell
without further transformation leading to block ATP
production via glycolysis.
Up-to-date anticancer activity of DCA and 2DG has
been demonstrated against many types of malignant
cells in vitro and in vivo [3, 5–7], and both compounds
are being tested in clinical trials of phases І–ІІ [8, 9].
However, so far, the use of these compounds as single
anticancer agents did not show apparently positive
results and their effectiveness in experimental tumors
was insufficient [10–12]. Therefore is seems reason-
able to study these compounds in combination with
other antitumor agents to enhance their anticancer
efficacy and to decrease their toxicity; such approach
has shown promising results [13].
The aim of present study was to analyze antitumor
and antimetastatic activity of DCA combined with 2DG
against Lewis lung carcinoma (LLC).
MATERIALS AND METHODS
Experimental animals and tumor cells. The
study was carried out on С57Bl/6 mice 2–2.5 months
Submitted: July 15, 2016.
*Correspondence: E-mail: pyaskovskaya@gmail.com
Abbreviations used: 2DG — 2-deoxy-D-glucose; DCA — dichloro-
acetate; EPR — electron paramagnetic resonance; LDH — lactate
dehydrogenase; LLC/R9 — high angiogenic variant of Lewis lung
carcinoma; MtETC — mitochondrial electron transport chain;
PDH — pyruvate dehydrogenase; PDK — pyruvate dehydrogenase
kinase; ROS — reactive oxygen species.
Exp Oncol 2016
38, 3, 176–180
Experimental Oncology 38, 176–180, 2016 (September)38, 176–180, 2016 (September) (September) 177
old weighting 18.5–21.5 g, bred at animal facility
of R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology (IEPOR, Kyiv, Ukraine).
The use and care of the experimental animals have
been performed in accordance with the standard in-
ternational rules of biologic ethics and was approved
by Institutional Animal Care and Use Committee.
As experimental tumor model, we have used LLC
variant LLC/R9, resistant to cisplatin and generated
from the parental LLC strain by multiple sequential
cycles of cisplatin administration to tumor-bearing
mice in vivo [14]. LLC/R9 cells were maintained in vitro
in RPMI 1640 culture medium (Sigma, USA) supple-
mented with 10% fetal bovine serum (FBS) (Sigma,
USA) and 40 µg/ml gentamycin at 37 °С in humidified
atmosphere with 5% СО2.
For in vivo experiments, LLC/R9 cells were propa-
gated in vitro at standard conditions and inoculated
i.m. to the mice (106 cells in 0.1 ml of Hank’s solution
per animal).
The number of cells in suspension and their viability
were routinely evaluated using hemocytometer and
0.4% trypan blue solution.
Agents, dosages and administration sche
dule. DCA (Sigma, USA) and 2DG (Sigma, USA)
were admini stered per os at total doses of 1.5 and
0.98 g/kg, respectively (both doses corresponded
to therapeutical range and were well tolerated [15–17])
at the regimens of a single use or combined treatment.
After tumor transplantation, experimental animals
were randomly distributed in four groups: group 1 —
mice treated with DCA (n = 11), group 2 — mice treated
with 2DG (n = 13), group 3 — mice treated with DCA
in combination with 2DG (n = 13). In the last case, 2DG
was administered at least 3 h after DCA. Mice treated
with water at the same regimen and in the same volume
served as the control (n = 12).
Treatment with DCA and 2DG has been initiated
on the following day after tumor cell transplantation,
5 times per week for 3 weeks. DCA and 2DG solutions
were prepared ex tempore in water, and were admin-
istered in a volume of 0.4 ml/animal.
Antitumor and antimetastatic activity of the
studied compounds were evaluated on day 21 after
tumor transplantation. Primary tumor diameter was
measured triply per week starting from the moment
of development of palpable tumors. Tumor volume (V)
was calculated by the formula:
V = 0.52d3,
where d — tumor diameter.
Anticancer activity was evaluated as tumor growth
inhibition rate calculated by the formula:
[(Vk – V) / Vk] • 100%,
where V, Vk — average tumor volumes in experi-
mental and control groups, respectively.
The number and volume of lung metastases was
routinely analyzed using binocular microscope and
millimeter scale.
Volume of metastases (V) was calculated by the
formula:
V =
1
∑
N
π di
3 / [ni × 6],
where ni — number of metastases with the dia-
meter of di.
Functional state of the components of mito
chondrial respiratory chain of tumor cells was ana-
lyzed using electron paramagnetic resonance (EPR).
Tumor tissue was cut into the samples of cylindrical
shape (d = 4.0 mm, l = 25–35 mm), frozen and stored
at −70 °C. EPR analysis of the samples was performed
at 77 К using spectrometer Е-109 Varian (USA) at po-
tential sweep speed of 500 Е/min, modulation am-
plitude of 1.25•10 Е, power of super high frequency
radiation of 10.0 mW, constant session of apparatus
of 1.0 s. The levels of reduced nonheme iron-sulfur
(Fe-S) centers (g = 1.94) of MtETC proteins, nitrosyl
(NO) complexes of heme iron (gmed = 2.007) and non-
heme iron (gmed = 2.03) were determined by the data
of EPR spectra.
Contents of glucose, creatinine and urea
in blood plasma samples were determined with the use
of semiautomatic biochemical analyzer. Blood plasma
was prepared using heparinized tubes and stored at
–20 °С until the time of analysis.
Glucose content in tumor tissue homogenates
was determined by enzyme glucose-oxidant method
using the kit for glucose analysis in biologic fluids
(Sigma, USA) according to instructions of the manu-
facturer.
Lactate and pyruvate content in tumor tissue
homogenates was determined by enzyme spectro-
photometry method using lactate dehydrogenase
(Sigma, USA) [18].
The samples for biochemical study were collected
from 4–5 animals per group, frozen and stored at
–20 °С (blood plasma) or in liquid nitrogen (tumor tis-
sue) until the moment of analysis.
Total activity of lactate dehydrogenase (LDH)
in tumor tissue homogenates was determined
by the rate of NADH (Sigma, USA) oxidation registered
by spectrophotometry by the decrease of optical den-
sity at the wavelength of 340 nm [18].
Engulfing capacity of tumorassociated СD14+
cells was determined by flow cytometry with the use
of FITC-labeled staphylococcus.
Tumorassociated СD14+ cells were identi-
fied with the use of anti-СD14 antibodies (Becton
Dickinson, USA) according to the instructions of the
manufacturer [19].
Production of reactive oxygen species (ROS)
in tumor-associated СD14+ cells was determined by spec-
trofluorometry with the use of 2.7-dichlorofluorescein
diacetate (Sigma, USA) [19].
Statistical analysis of the data was performed
by descriptive statistics, Student’s t-test and Mann —
Whitney U test, with the use of Microsoft Excel and
Microcal Origin programs.
RESULTS AND DISCUSSION
It has been shown that combination of DCA and
2DG exerted significant antitumor activity, while both
178 Experimental Oncology 38, 176–180, 2016 (September)
studied compounds as single agents were practically
inactive in inhibiting primary tumor growth. The data
on the influence of DCA, 2DG and their combination
on the primary tumor growth and metastasis at 21st
day after LLC transplantation are presented in Fig. 1.
As one may see, at this time in LLC/R9-bearing mice
treated with DCA or 2DG the primary tumor volume
did not differ significantly from that in control group
(see Fig. 1). Meanwhile, DCA combined with 2DG
significantly inhibited tumor growth starting from the
16th day and on the 21st day tumor volume was by 70%
(p < 0.05) lower compared to the control.
0
20
40
60
80
100
120
140
160
180
Tumor volume Metastases number Metastases volume
Tu
m
or
a
nd
m
et
as
ta
si
s
in
di
ce
(
%
)
Control
DCA
2DG
DCA+2DG
*
*
*
*
*
Fig. 1. Effect of DCA, 2DG and their combination on tumor
growth and metastasis indices in LLC/R9 bearing mice.
*р < 0.05 as compared to the control
In regard to antimetastatic effects, only DCA but
not 2DG suppressed metastasizing. In animals treated
with DCA the number and volume of lung metastases
decreased nearly by 60% (р < 0.05) and more than
by 90% (р < 0.05), respectively, as compared to the
control indexes. Meanwhile, no antimetastatic activity
was revealed in 2DG treated mice (see Fig. 1). In the
case of combined treatment of animals with 2DG and
DCA antimetastatic effects were related to activity
of DCA only, but not 2DG: in this group of mice the
number and volume of lung metastases decreased
by 46% (р < 0.05) and nearly by 90% (р < 0.05), re-
spectively, compared to the control group.
The study of lactate and pyruvate levels in the tu-
mor tissue has shown that antitumor activity of DCA
combined with 2DG was mediated by their significant
effect on glucose metabolism reflected in decreased
lactate level and accumulation of pyruvate in the tumor.
As shown in Fig. 2, combined treatment of mice with
DCA and 2DG resulted in 31% decrease (p < 0.05)
of lactate content and 48% increase (p < 0.05) of py-
ruvate content compared to the control values. Such
effects could reflect the decreased utilization of pyru-
vate in the tricarboxylic acid cycle and/or decreased
LDH activity in the reactions of pyruvate to lactate
reduction. As far as LDH activity did not change sig-
nificantly upon combined use of DCA and 2DG (Fig. 2),
accumulation of pyruvate could be related to the de-
creased intensity of the reactions initiated by pyruvate
as a substrate. This could be accompanied not only
with pyruvate accumulation, but also with an increased
pool of NAD-H and the decreased pool of NAD+ which
is an electron acceptor providing glycolysis function-
ing. It couldn’t be excluded that altered level of lactate
and pyruvate in tumor tissue may be related to the
competition between 2DG and glucose.
0
50
100
150
200
Lactate Pyruvate LDH
*
* *
In
di
ce
(%
)
Control
DCA
2DG
DCA + 2DG
Fig. 2. Effect of DCA, 2DG and their combination on lactate
and pyruvate content and LDH activity in tumor tissue of LLC/
R9 bearing mice. *р < 0.05 as compared to the control
In the group of animals treated with DCA and 2DG,
lactate content decreased not only in tumor tissue but
also in blood plasma (by 38% compared to the control,
p < 0.05, Table 1). The decrease of lactate content
in blood plasma close to 30% (p < 0.05) was also
observed in the group of mice treated with 2DG only
indicating the contribution of 2DG into the decrease
of lactate content in the tumor and blood plasma of ani-
mals treated by 2DG and DCA combination.
Table 1. Effect of DCA, 2DG and their combination on blood plasma bio-
chemical indices of LLC/R9 bearing mice
Group of mice Glucose, mM Lactate, mM Creatinine,
mM Urea, mM
Control 5.3 ± 0.9 8.5 ± 0.9 31.8 ± 1.5 4.5 ± 0.5
DCA 5.7 ± 0.8 8.2 ± 0.5 38.3 ± 2.0* 3.6 ± 0.4
2DG 6.5 ± 0.3 6.2 ± 0.5* 35.4 ± 1.0 4.2 ± 0.5
DCA + 2DG 4.8 ± 1.1 5.2 ± 0.7* 33.3 ± 4.5 3.1 ± 0.6
Note: *р < 0.05 as compared to the control.
As shown in Table 1, decreased lactate content
in blood plasma of mice treated with 2DG or 2DG com-
bined with DCA, did not affect glucose content in blood
plasma. At the same time, in animals treated with DCA
we have registered significant increase of creatinine
content (р < 0.05) suggesting an ability of this com-
pound to cause kidney malfunction in tumor-bearing
animals.
Earlier we have shown that high antitumor efficacy
of DCA, alone or in combination with antiangiogenic
aconitin-containing agent, against solid variant of Ehr-
lich carcinoma was related to the increased produc-
tion of ROS in СD14+ cells infiltrating the tumor with
increased nitrosylation of proteins of mitochondrial
ETC in tumor cells [20]. However, the studied agents
have no effect on the functional state of mitochondrial
ETC in LLC cells and nitrosylation levels of proteins
of mitochondrial ETC. As one may see in Fig. 3, in LLC/
R9 bearing animals treated with DCA combined with
2DG or DCA only, an intensity of EPR signals cor-
responding to NO-complexes of heme (gmed = 2.007)
or nonheme (gmed = 2.03) iron in proteins of mitochon-
drial ETC were not statistically higher than analogous
indexes in the control. Similar pattern has been ob-
served for EPR signal intensity of Fe-S cluster proteins
Experimental Oncology 38, 176–180, 2016 (September)38, 176–180, 2016 (September) (September) 179
(g = 1.94), which, as it is known, characterize functional
activity of ETC complex I: in all groups of animals this
index was practically equal to that in the control.
Significant increase (р < 0.05) of nitrosylation level
of heme and nonheme proteins has been registered
only in the group of animals treated with 2DG, that may
be attributed to a capability of this compound to modify
the production of reactive nitrogen species [21]. How-
ever, an enhancement of antitumor activity of DCA with
2DG was not related to its influence on mitochondrial
ETC functioning in LLC cells.
An analysis of a functional state of tumor-infiltrating
CD14+-macrophage like cells has shown that antitumor
effect of DCA in combination with 2DG was realized,
at least in part, via enhancement of their cytotoxic ac-
tivity. In the group of animals treated with DCA in com-
bination with 2DG, the level of ROS production in these
cells increased by 120% (p < 0.01) compared with that
in the control (Fig. 4). In these groups of animals, the
relative number of tumor-infiltrating CD14+ cells and
their engulfing capacity did not differ significantly from
corresponding indexes in the control. It is necessary
to note that in the groups of mice treated with DCA
or 2DG the number and functional activity of CD14+
cells did not differ significantly from control group.
0
50
100
150
200
250
NO-nonheme NO-heme Fe-S
EP
R
si
gn
al
(%
)
Control
DCA
2DG
DCA + 2DG*
*
Fig. 3. Effect of DCA, 2DG and their combination on the indices
of functional state of MtETC in the tumor cells. *р < 0.05 as com-
pared to the control
0
50
100
150
200
250
Cell number Engulfing capacity ROS
*
In
di
ce
s
(%
)
Control
DCA
2DG
DCA + 2DG
Fig. 4. Effect of DCA, 2DG and their combination on the indices
of functional state of tumor-infiltrating CD14+ macrophage like
cells. *р < 0.05 as compared to the control
Therefore, the present work has demonstrated
significant enhancement of antitumor action of DCA
used in combination with other inhibitor of energetic
metabolism, 2DG, even in the absence of antitumor
effects of these agents used at the same dosages
in monotherapy mode. According to literature data,
antitumor activity of DCA is largely based on its capa-
bility for negative regulation of PDK, a negative regula-
tor of enzymes of mitochondrial PDH complex, which
plays a key role in regulation of tricarboxylic acid cycle
and oxidative phosphorylation [22]. In the case of PDK
inhibition with DCA, tumor cell metabolism switches
from glycolysis to oxidative phosphorylation that
should affect lactate production in tumor tissue. In our
work, antitumor effectiveness of DCA in combination
with 2DG was associated with inhibition of glycolysis,
as it has been illustrated by the drop of lactate content
in the tumor. However, the relation between antitumor
activity of DCA and 2DG and their influence on tumor
metabolism has been found only in the case of their
combined use, supposedly due to the enhancement
of pharmacological effects of these compounds.
In other words, an effective inhibition of glycolysis in tu-
mor cells sufficient for realization of antitumor action
has been exerted as a consequence of simultaneous
intensification of oxidative phosphorylation in tumor
cells via DCA-induced activation of PDH complex and
competitive inhibition of glucose consumption by tu-
mor cells by 2DG, and consequently, decreased en-
ergy and/or concentration of glycolytic intermediates.
Antitumor effectiveness of DCA combined with
2DG could be related not only to glycolysis inhibition,
but also to their effect on tumor microenvironment,
namely, activation of tumor-infiltrating cells expressing
CD14 receptor [23]. Capability of DCA to affect dif-
ferent types of immunocompetent cells, in particular,
to cause alternative activation of tumor-associated
macrophages has been shown in several studies [24,
25]. Our data evidenced on the capability of the studied
agents to increase cytotoxic activity of tumor infiltrating
CD14+ immunocompetent cells (reflected in increased
intracellular production of ROS in these cells), while
there was no influence of phagocytic (engulfing) ac-
tivity of the cells. Such effect on the functional state
of CD14+ cells was observed only in the case of the
combined use of DCA and 2DG.
At last, one should note that while DCA-based
monotherapy was found to be insufficient for achieve-
ment of antitumor effect, DCA, but not 2DG, exerted
an expressed antimetastatic activity against LLC/
R9. Combined use of DCA and 2DG did not enhance
such antimetastatic effect. Antimetastatic acti vity
of DCA has been demonstrated in other studies,
in particular, against rat mammary adenocarcinoma
cells if administered by intravenous route [26]. Pos-
sible mechanism of antimetastatic activity of DCA was
discussed in the study [27], where it has been shown
that activation of mitochondrial PDH complex in tumor
cells significantly increased their sensitivity to anoikis
and, correspondingly, decreased their metastatic
potential. Taking into account high antimetastatic ac-
tivity of DCA against LLC/R9, capability of this agent
to promote activation of PDH complex through PDK
inhibition in tumor cells, and the data on the close re-
lation between activity of PDH complex in tumor cells
180 Experimental Oncology 38, 176–180, 2016 (September)
and their metastatic potential, one could suppose that
DCA could be considered as a promising agent with
high antimetastatic activity.
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