Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis
A hallmark of malignancy is excessive tumor glycolysis, even in the presence of oxygen, which causes lactacidosis in the tumor microenvironment and favors tumor cell proliferation and survival. For this reason antimetabolic agents which target tumor cell metabolism are being researched extensively a...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis / D.L. Kolesnik, O.N. Pyaskovskaya, I.V. Boychuk, O.I. Dasyukevich, O.R. Melnikov, A.S. Tarasov, G.I. Solyanik // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 126-129. — Бібліогр.: 16 назв. — англ. |
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Kolesnik, D.L. Pyaskovskaya, O.N. Boychuk, I.V. Dasyukevich, O.I. Melnikov, O.R. Tarasov, A.S. Solyanik, G.I. 2019-01-22T11:34:29Z 2019-01-22T11:34:29Z 2015 Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis / D.L. Kolesnik, O.N. Pyaskovskaya, I.V. Boychuk, O.I. Dasyukevich, O.R. Melnikov, A.S. Tarasov, G.I. Solyanik // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 126-129. — Бібліогр.: 16 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145468 A hallmark of malignancy is excessive tumor glycolysis, even in the presence of oxygen, which causes lactacidosis in the tumor microenvironment and favors tumor cell proliferation and survival. For this reason antimetabolic agents which target tumor cell metabolism are being researched extensively as promising anticancer drugs. Aim: To study the effect of lactacidosis on survival of Lewis lung carcinoma (LLC) cells at the conditions of nutritional substrate deficiency in vitro and evaluate antitumor and antimetastatic activity against LLC/R9 in vivo. Materials and Methods: LLC variant LLC/R9 was used as experimental tumor model. Tumor cell viability was determined using trypan blue staining. Apoptosis level was counted with the use of Hoechst 33258 dye. Lactate content in the tumor tissue was evaluated by enzyme method with the use of lactate dehydrogenase. Reactive oxygen species was determined using 2.7-dichlorofluorescein diacetate. Effects of dichloroacetate (DCA) on the growth and metastasis of LLC/R9 were analyzed by routine procedures. Evaluation of DCA effect toward electron-transport chain (ETC) components was performed using EPR. Results: It has been shown that at the conditions of lactacidosis and glucose deficiency, LLC/R9 cell viability in vitro was higher by 30% (р < 0.05) and apoptosis level was triply lower (р < 0.05) than these indices at the conditions of glucose deficiency only. In mice with transplanted LLC/R9 tumors treated for 3 weeks per os with DCA at the total dose of 1.5 g/kg of body weight starting from the next day after tumor transplantation, the primary tumor volume was just by 30% lower than that in control group. At the same time, the number and volume of lung metastases in animals treated with DCA were by 59% (р < 0.05) and 94% (р < 0.05) lower, respectively, than these indices in the control group. DCA treatment resulted in nearly 30% increase (р < 0.05) of lactate content in tumor tissue compared to that in the control, but did not affect significantly the levels of heme iron complexes with NO (at gmed = 2.007) in mitochondrial ETC proteins and Fe-S cluster proteins (at g = 1.94) in tumor cells. Conclusions: It has been shown that lactacidosis significantly promoted LLC/R9 cell survival at the conditions of glucose deficiency in vitro. If LLC/R9 developed in vivo, DCA as the compound with antilactacidosis activity did not suppress significantly the primary tumor growth but exerted significant antimetastatic activity. Key Words: dichloroacetate, Lewis lung carcinoma, lactacidosis. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis |
| spellingShingle |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis Kolesnik, D.L. Pyaskovskaya, O.N. Boychuk, I.V. Dasyukevich, O.I. Melnikov, O.R. Tarasov, A.S. Solyanik, G.I. Original contributions |
| title_short |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis |
| title_full |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis |
| title_fullStr |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis |
| title_full_unstemmed |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis |
| title_sort |
effect of dichloroacetate on lewis lung carcinoma growth and metastasis |
| author |
Kolesnik, D.L. Pyaskovskaya, O.N. Boychuk, I.V. Dasyukevich, O.I. Melnikov, O.R. Tarasov, A.S. Solyanik, G.I. |
| author_facet |
Kolesnik, D.L. Pyaskovskaya, O.N. Boychuk, I.V. Dasyukevich, O.I. Melnikov, O.R. Tarasov, A.S. Solyanik, G.I. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2015 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
A hallmark of malignancy is excessive tumor glycolysis, even in the presence of oxygen, which causes lactacidosis in the tumor microenvironment and favors tumor cell proliferation and survival. For this reason antimetabolic agents which target tumor cell metabolism are being researched extensively as promising anticancer drugs. Aim: To study the effect of lactacidosis on survival of Lewis lung carcinoma (LLC) cells at the conditions of nutritional substrate deficiency in vitro and evaluate antitumor and antimetastatic activity against LLC/R9 in vivo. Materials and Methods: LLC variant LLC/R9 was used as experimental tumor model. Tumor cell viability was determined using trypan blue staining. Apoptosis level was counted with the use of Hoechst 33258 dye. Lactate content in the tumor tissue was evaluated by enzyme method with the use of lactate dehydrogenase. Reactive oxygen species was determined using 2.7-dichlorofluorescein diacetate. Effects of dichloroacetate (DCA) on the growth and metastasis of LLC/R9 were analyzed by routine procedures. Evaluation of DCA effect toward electron-transport chain (ETC) components was performed using EPR. Results: It has been shown that at the conditions of lactacidosis and glucose deficiency, LLC/R9 cell viability in vitro was higher by 30% (р < 0.05) and apoptosis level was triply lower (р < 0.05) than these indices at the conditions of glucose deficiency only. In mice with transplanted LLC/R9 tumors treated for 3 weeks per os with DCA at the total dose of 1.5 g/kg of body weight starting from the next day after tumor transplantation, the primary tumor volume was just by 30% lower than that in control group. At the same time, the number and volume of lung metastases in animals treated with DCA were by 59% (р < 0.05) and 94% (р < 0.05) lower, respectively, than these indices in the control group. DCA treatment resulted in nearly 30% increase (р < 0.05) of lactate content in tumor tissue compared to that in the control, but did not affect significantly the levels of heme iron complexes with NO (at gmed = 2.007) in mitochondrial ETC proteins and Fe-S cluster proteins (at g = 1.94) in tumor cells. Conclusions: It has been shown that lactacidosis significantly promoted LLC/R9 cell survival at the conditions of glucose deficiency in vitro. If LLC/R9 developed in vivo, DCA as the compound with antilactacidosis activity did not suppress significantly the primary tumor growth but exerted significant antimetastatic activity. Key Words: dichloroacetate, Lewis lung carcinoma, lactacidosis.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/145468 |
| citation_txt |
Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis / D.L. Kolesnik, O.N. Pyaskovskaya, I.V. Boychuk, O.I. Dasyukevich, O.R. Melnikov, A.S. Tarasov, G.I. Solyanik // Experimental Oncology. — 2015. — Т. 37, № 2. — С. 126-129. — Бібліогр.: 16 назв. — англ. |
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| fulltext |
126 Experimental Oncology 37, 126–129, 2015 (June)
EFFECT OF DICHLOROACETATE ON LEWIS LUNG CARCINOMA
GROWTH AND METASTASIS
D.L. Kolesnik*, O.N. Pyaskovskaya, I.V. Boychuk, O.I. Dasyukevich, O.R. Melnikov, A.S. Tarasov, G.I. Solyanik
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology,
NAS of Ukraine, Kyiv 03022, Ukraine
A hallmark of malignancy is excessive tumor glycolysis, even in the presence of oxygen, which causes lactacidosis in the tumor microen-
vironment and favors tumor cell proliferation and survival. For this reason antimetabolic agents which target tumor cell metabolism are
being researched extensively as promising anticancer drugs. Aim: To study the effect of lactacidosis on survival of Lewis lung carcinoma
(LLC) cells at the conditions of nutritional substrate deficiency in vitro and evaluate antitumor and antimetastatic activity against LLC/
R9 in vivo. Materials and Methods: LLC variant LLC/R9 was used as experimental tumor model. Tumor cell viability was determined
using trypan blue staining. Apoptosis level was counted with the use of Hoechst 33258 dye. Lactate content in the tumor tissue was
evaluated by enzyme method with the use of lactate dehydrogenase. Reactive oxygen species was determined using 2.7-dichlorofluorescein
diacetate. Effects of dichloroacetate (DCA) on the growth and metastasis of LLC/R9 were analyzed by routine procedures. Evaluation
of DCA effect toward electron-transport chain (ETC) components was performed using EPR. Results: It has been shown that at the
conditions of lactacidosis and glucose deficiency, LLC/R9 cell viability in vitro was higher by 30% (р < 0.05) and apoptosis level was
triply lower (р < 0.05) than these indices at the conditions of glucose deficiency only. In mice with transplanted LLC/R9 tumors treated
for 3 weeks per os with DCA at the total dose of 1.5 g/kg of body weight starting from the next day after tumor transplantation, the pri-
mary tumor volume was just by 30% lower than that in control group. At the same time, the number and volume of lung metastases
in animals treated with DCA were by 59% (р < 0.05) and 94% (р < 0.05) lower, respectively, than these indices in the control group. DCA
treatment resulted in nearly 30% increase (р < 0.05) of lactate content in tumor tissue compared to that in the control, but did not affect
significantly the levels of heme iron complexes with NO (at gmed = 2.007) in mitochondrial ETC proteins and Fe-S cluster proteins (at g
= 1.94) in tumor cells. Conclusions: It has been shown that lactacidosis significantly promoted LLC/R9 cell survival at the conditions
of glucose deficiency in vitro. If LLC/R9 developed in vivo, DCA as the compound with antilactacidosis activity did not suppress signifi-
cantly the primary tumor growth but exerted significant antimetastatic activity.
Key Words: dichloroacetate, Lewis lung carcinoma, lactacidosis.
It is well known that lactacidosis, large accumulation
of lactate and decrease of рН, is the main characteris-
tics of metabolic tumor cell microenvironment in vitro
and in vivo. Earlier lactacidosis has been considered
as a ballast product of tumor cell metabolism. However,
recently it has been shown that it could be used by tu-
mor cells as an effective energetic fuel and be among
the factors responsible for tumor resistance to glucose
deficiency [1–3]. As we have shown using Lewis lung
carcinoma (LLC)/R9 cells, LLC variant sensitive to an-
tiangiogenic cancer therapy [4, 5], lactacidosis could
promote tumor cell survival at the conditions of nutri-
tional deficiency. Such conditions were generated via
long-term incubation of tumor cells without replace-
ment of culture medium (“unfed culture” model) [6].
The study of tumor cell growth kinetics at the conditions
of “unfed culture” has demonstrated that at the back-
ground of complete absence of glucose in incubation
medium at days 7–8 of cell growth the viable cell counts
did not fall below the third part from their maximum
registered at days 3–4, and remained practically at this
level till the 10th day. High survival rate of LLC/R9 cells
at the conditions of “unfed culture” was related in par-
ticular to the ability of these cells to macroautophagy.
However, it could not be excluded that the ability of LLC/
R9 cells for adaptation toward nutritional substrate defi-
ciency was determined by lactacidosis which developed
as a consequence of prolonged tumor cell culturing
without replacement of incubation medium.
If lactacidosis is capable to increase tumor cell
survival, then the compounds suppressing lactacidosis
formation in tumor microenvironment, in particular, di-
chloroacetate (DCA) as a compound with antilactacidosis
activity should demonstrate antitumor activity. The pre-
sent study was aimed on the testing of such assumption.
It is known that DCA is an inhibitor of pyruvate de-
hydrogenase kinase (PDK), that’s why it is considered
as a negative regulator of enzymes of mitochondrial py-
ruvate dehydrogenase (PDH) complex which plays a key
role in the regulation of tricarboxylic acid and oxidative
phosphorylation [7]. If PDH complex is phosphorylated,
an entry of pyruvate in Krebs cycle is inhibited, so gly-
colysis is being activated. Due to PDK inhibition, DCA
is capable to lead for indirect activation of PDH com-
plex enzymes and respectively causes the shift of cell
energetic balance from glycolysis toward activation
of oxidative phosphorylation. Therefore, DCA has been
widely used for correction of lacticemia caused by high
intensity of glycolysis or defective cell respiration.
According to the data of literature, an ability of DCA
to activate oxidative phosphorylation underlies its
antitumor activity and is realized, in particular, via
the decrease of lactacidosis and induction of reactive
oxygen species (ROS) [8–12]. The aim of our study
was to analyze an influence of lactacidosis on survival
Submitted: March 20, 2015.
*Correspondence: E-mail: deniskol@mail.ru
Abbreviations used: DCA — dichloroacetate, ETC — electron-trans-
port chain; LLC/R9 — Lewis lung carcinoma variant; PDH — pyru-
vate dehydrogenase; PDK — pyruvate dehydrogenase kinase.
Exp Oncol 2015
37, 2, 126–129
Experimental Oncology 37, 126–129, 2015 (June) 127
of LLC/R9 cells at the conditions of nutrient deficiency
in vitro and evaluate antitumor and antimetastatic acti-
vity of DCA against LLC/R9 in vivo.
MATERIALS AND METHODS
Experimental animals, tumor cells. The study
was carried out using 2.0–2.5 months old C57Bl/6 mice
weighting 18–23 g, bred at animal facility of R.E. Ka-
vetsky Institute of Experimental Pathology, Oncology
and Radiobiology of the NAS of Ukraine. Animal study
protocols and operation procedures were carried out
in accordance with the main requirements to keeping
and working with laboratory animals and to the rules
of local Bioethics Committee.
In the study LLC variant LLC/R9 derived from wild-
type LLC strain by 9 sequential chemotherapy in vivo
sessions based on cis-diamminedichloroplatinum (cis-
DDP), was used [13]. LLC/R9 cells were maintained
in RPMI culture medium (Sigma, USA) supplemented
with 10% fetal calf serum (FCS) (Sigma, USA), and
40 g/ml gentamycine at 37 °C in humidified atmo-
sphere with 5% CO2.
Experiments in vitro. Cell counts in suspension
and their viability was routinely analyzed on a hemo-
cytometer using trypan blue exclusion test.
For evaluation of effects of lactacidosis on viabi lity
of LLC/R9 cells, 1.5•105 cells/well were seeded in 24-
well plate in RPMI 1640 medium (Sigma, USA) with
standard glucose content. After overnight incubation
cell incubation medium was replaced with the fresh
media with different content of glucose, lactate and
with different pH for simulation of the conditions
of glucose deficiency, lactacidosis at the background
of glucose deficiency, as well as standard (Table 1).
Glucose deficient medium was prepared on the basis
of RPMI 1640 medium without glucose (Sigma, USA).
Lactacidosis was generated by adding pure lactic acid
(Sigma, USA) to glucose deficient medium to a final
concentration of 14 ± 0.7 mM and pH 6.7.
Table 1. The content of glucose, lactate and pH of culture media used
in the study
Medium Glucose content,
mM
Lactate content,
mM рН
Standard 9.0 ± 0.5 1.6 ± 0.1 7.4 ± 0.01
Glucose deficiency 3.0 ± 0.1 1.6 ± 0.1 7.4 ± 0.01
Lactacidosis 3.0 ± 0.1 14.0 ± 0.7 6.7 ± 0.01
Effect of different incubation conditions upon tu-
mor cell survival, ROS production, glucose consump-
tion and lactate production were estimated on the 2nd
day of tumor cell incubation.
Glucose content in culture media and 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 manufacturer. Lactate content
in incubation media and in tumor tissue homogenates
was determined by enzyme spectrophotometry me-
thod using lactate dehydrogenase (Sigma, USA) [14].
Medium and tumor tissues samples were collected and
stored at −20 °С or in liquid nitrogen, correspondently,
until the measurement performance.
Apoptosis level in tumor cells was analyzed with
the use of Hoechst 33258 dye (Sigma, USA) and fluo-
rescent microscopy by standard method.
Production of ROS in tumor cells was determined
with the use of 2.7-dichlorofluorescein diacetate (Sig-
ma, USA) by spectrofluorometry (excitation at 495 nm,
emission at 530 nm) according to [15].
All measurements were repeated.
Experiments in vivo. For in vivo experiments,
LLC/R9 cells were propagated in vitro at standard con-
ditions and were inoculated i.m. to mice (1.0•106 cell/
animal in 0.1 ml of Hanks’ solution).
After LLC/R9 cell inoculation the animals were dis-
tributed into 2 groups: group 1 — mice treated with DCA
(Sigma, USA) at the total dose of 1.5 g/kg (LD50/3)
(n = 13); group 2 — mice treated with water at the same
regimen and in the same volume (control, n = 12).
The treatment has been initiated on the following
day after tumor cell transplantation at metronomic
regimen, 5 times per week for 3 weeks. DCA was
prepared ex tempore in water, and was administered
per os in a volume of 0.4 ml/animal.
Primary tumor volume was calculated on the basis
of its diameter measured using caliper each 3rd day
starting from the 10th day after tumor cell inoculation,
by the formula:
V = π (d)3/6,
where d — diameter of tumor (mm).
Metastasis level in tumor bearing mice was
evaluated at the 21st day after tumor cell inoculation
by the number and volume of lung metastases using
binocular microscope and millimeter scale.
Total volume of metastasis was calculated
by the formula:
V = Σ ni π(di)3/6,
where V — total volume of metastases (mm3), ni —
number of metastases with the diameter of di (mm).
Analysis of functional activity of mitochondrial
respiratory chain components in tumor cells was per-
formed with the use of EPR at the 21st day after tumor
cell inoculation. Tumor tissue was cut into the samples
of special size (d = 4.0 mm, l = 25–35 mm), frozen and
stored at −70 °C. EPR analysis of the samples was per-
formed at 77 К using spectrophotometer Е-109 Var-
ian (USA) at potential sweep speed of 500 Е/min,
modulation amplitude of 1.25×10 Е, power of SHF-
irradiation of 10.0 mW, constant session of apparatus
of 1.0 s. The levels of heme iron complexes with NO
(at gmed = 2.007) in mitochondrial ETC proteins and
Fe-S cluster proteins (at g = 1.94) in tumor cells were
determined by the data of EPR spectra.
Statistical analysis of obtained results was car-
ried out by descriptive methods, nonlinear regression
analysis and Student’s t-test with the use of Microsoft
Excel and Microcal Origin programs.
RESULTS
It has been shown that lactacidosis at the condi-
tions of glucose deficiency significantly promoted
survival of LLC/R9 cells. Indeed, growth kinetics of tu-
128 Experimental Oncology 37, 126–129, 2015 (June)
mor cells incubated at the conditions of lactacidosis
at the background of glucose deficiency did not differ
significantly of that of the cells incubated in the me-
dium with standard glucose content, at least at the pe-
riod of their exponential growth. In particular, viable cell
counts at the 2nd day of incubation at the conditions
of lactacidosis at the background of glucose deficiency
was practically the same as in the case of cell incuba-
tion in the medium with standard glucose content.
At the same time, in both cases (lactacidosis and
standard) the viable cell counts were nearly by higher
30% (р < 0.05) than in the case of cell incubation
at the conditions of glucose deficiency itself (Fig. 1, a).
Apart from this, the number of apoptotic cells
at the conditions of lactacidosis also did not differ
statistically from that index in the case of cell incuba-
tion in the medium with standard glucose content and
at the 2nd day was equal to 8.5 ± 0.9%, while at the con-
ditions of glucose deficiency the number of apoptotic
cells was nearly three times higher (p < 0.05) than that
in the case of lactacidosis (Fig. 1, b).
0
20
40
60
80
100
120
1 2
*
3
Culture conditions
Nu
m
be
r o
f v
ia
bl
e
ce
lls
, %
0
50
100
150
200
250
1 2
*
3
Culture conditions
Nu
m
be
r o
f a
po
pt
ot
ic
c
el
ls
, %
a b
Fig. 1. LLC/R9 cell survival on the 2nd day of their incuba-
tion in standard (1), glucose deficient (2) and lactacidosis (3)
media; a — the number of viable cells; b — apoptosis level.
*p < 0.05 compared to control
Interestingly, at the conditions of lactacidosis
in LLC/R9 cells glucose consumption was significantly
lower. Low rate of glucose consumption by tumor cells
upon lactacidosis registered just at 1st day of their in-
cubation, has been restored at 2nd day and was by 70%
lower (p < 0.05) than that in the case of the medium with
standard glucose content (Table 2). In the case of glu-
cose deficiency in contrary to lactacidosis at 2nd day the
level of glucose in incubation medium fall to zero, what
additionally evidenced on decreased glucose consump-
tion by LLC/R9 cells at the conditions of lactacidosis.
While lactacidosis led to decreased rate of glucose
intake by LLC/R9 cells, the level of intracellular ROS
in the cells that survived at such conditions significantly
increased. These data are presented in Table 2 and
demonstrate that ROS level in the cells incubated
at the conditions of lactacidosis was nearly by 150%
(p < 0.05) and 230% (p < 0.05) higher than corre-
sponding indices for the cells incubated in standard
and glucose-deficient media, respectively.
So, the obtained data have demonstrated that
lactacidosis significantly promoted LLC/R9 cell sur-
vival at the conditions of glucose deficiency in vitro
what is supported by high counts of the cells survived
at such unfavorable conditions, and by low apoptosis
rate. The cell survival was associated with unexpected
increase of intracellular ROS level and decreased glu-
cose consumption in LLC/R9.
Table 2. The effect of lactacidosis at the condition of the glucose deficiency
upon glucose consumption and ROS production by tumor cells in vitro
Medium Glucose consumption, % ROS, %
Standard 100.0 ± 5.9 100.0 ± 24.8
Glucose deficiency 0.0 ± 0.0* 75.8 ± 10.7
Lactacidosis 29.8 ± 1.5* 248.7 ± 53.2*
Note: *p < 0,05.
These patterns of survival of LLC/R9 cells
at the conditions of lactacidosis at the background
of glucose deficiency in vitro evidenced on the fact
that decreased lactate content in tumor microenviron-
ment may prevent tumor cell survival at the conditions
of metabolic stress therefore exerting antitumor effect.
Such hypothesis was tested by us with the use of DCA
as a compound capable to reduce lactacidosis.
The data on DCA influence on LLC/R9 growth
kinetics and metastasis are shown on Fig. 2 and
Table 3. According to these data, DCA did not affect
significantly the growth of primary tumors, but caused
an expressed suppression of metastasis. The growth
kinetics of primary tumors in mice with LLC/R9 treated
with DCA did not practically differ from that in control
mice, and at the 21st day after tumor transplantation
the volume of primary tumors in experimental group
was just by 39% lower than that in the control group
(see Fig. 2, Table 3). Despite the fact that DCA exerted
no notable suppression of primary tumor growth,
its antimetastatic activity toward LLC/R9 was found
to be striking. The number and volume of lung me-
tastases in tumor bearing mice treated with DCA were
by 59% (р < 0.05) and 94% (р < 0.05) lower than these
indices in control group, respectively (see Table 3).
0
400
800
1200
1600
2000
0 3 6 9 12 15 18 21
Day after tumor cell inoculation
Tu
m
or
v
ol
um
e,
m
m
3
Control
DCA
Fig. 2. The effect of DCA upon LLC/R9 growth kinetics in vivo
Table 3. Influence of DCA on LLC/R9 growth and metastasis
Group of mice Tumor volume,
mm3
Number of me-
tastasis
Volume of metastasis,
mm3
Control (n = 13) 1702.7 ± 333.9 10.9 ± 1.2 17.9 ± 5.6
DCA (n = 13) 1046.0 ± 258.3 4.5 ± 1.6* 1.1 ± 0.4*
Note: *р < 0.05, differences are significant as compared to the value for control.
An analysis of lactate content in tumor tissue
samples has demonstrated that unexpectedly DCA
caused significant increase of lactate content in tumor
tissue, at least at the 21st day after tumor transplan-
tation. As one may see in Table 4, lactate content
Experimental Oncology 37, 126–129, 2015 (June) 129
in tumor tissue of mice treated with DCA, was nearly
by 30% higher (р < 0.05) than that in the control.
Accounting an ability of DCA as PDH kinase inhibitor
reorganize energetic metabolism of malignant tumor
toward oxidative phosphorylation, we have consi dered
the lactate production by tumor cells as a surrogate
marker of glycolysis inhibition upon DCA influence.
The increase of lactate level in tumor caused by DCA
indicated that its administration to mice with LLC/
R9 at a total dose of 1.5 g/kg of animal body weight
could be insufficient for activation of oxidative phos-
phorylation in tumor cells what explains in part its low
efficacy against primary tumors.
Table 4. Influence of DCA on the lactate level in tumor tissue of LLC/R9-
bearing mice
Group of mice Lactate (μmol/1 g tissue)
Control (n = 4) 11.1 ± 0.6
DCA (n = 5) 14.4 ± 1.5*
Note: р < 0.05, differences are significant as compared to the value for control.
An analysis of EPR spectra of tumor samples has
shown that DCA did not affect significantly the functional
state of the ETC components in tumor cell mitochondria
(Table 5). For example, in mice with LLC/R9 treated
with DCA, an intensity of EPR signals corresponding
to nitrosyl-heme iron protein complexes (gсер = 2.007)
in ETC proteins of tumor cell mitochondria was not
significantly higher than that in control mice. It is known
that accumulation of NO-complexes of hem iron may
indicate from one side the redox imbalance toward
domination of free radical processes, in particular,
NO hyperproduction, and from other side on possible
inhibition of cell respiration via nitrosylation of heme
proteins. However, DCA, the main mechanism of an-
titumor action of which is thought to be related to the
induction of ROS production by mitochondria [8, 10,
11], did not cause elevation of heme iron complexes
with NO in tumor tissue. The latest observation could
be possibly related to the features of LLC/R9 cells,
namely, an extremely high content of these complexes
characteristic for this tumor and progressive accumula-
tion of which during tumor development in vivo has been
registered by us even in the absence of treatment [16].
Table 5. Influence of DCA on the mitochondrial ETC activity of tumor cells
Group of mice
Relative EPR signal intensity
Nitrosyl-heme iron protein
complexes (g = 2.007)
Fe-S protein
(g = 1.94)
Control 54.3 ± 4.5 15.8 ± 0.5
DCA 97.8 ± 30.1 17.8 ± 2.1
An absence of significant effect of DCA on func-
tional activity of mitochondrial ETC components in tu-
mor cells was also supported by the data on intensity
of EPR signals corresponding to Fe-S cluster proteins
(g = 1.94) (complexes І, ІІ, ІІІ), that was practically
equal in both animal groups (see Table 5).
In conclusion, the results of our study have shown
that lactacidosis significantly promoted the survival
of LLC variant LLC/R9 at the conditions of glucose de-
ficiency. At the same time, if LLC/R9 developed in vivo
DCA did not exert antitumor activity against primary
tumors. The failure of antitumor action of DCA against
LLC/R9 growth was in agreement with the absence
of DCA inhibition effect on lactate content in the tumor
as well as an absence of notable DCA effect on tumor
cell ROS production. Although DCA did not affect
LLC/R9 growth but drastically inhibited metastasis;
this observation could not be explained by DCA action
within primary tumor and further additional studies
of its antimetastatic action are required.
REFERENCES
1. Feron O. Pyruvate into lactate and back: from the Warburg
effect to symbiotic energy fuel exchange in cancer cells. Radio ther
Oncol 2009; 92: 329–33. doi: 10.1016/j.radonc.2009.06.025.
2. Wu H, Ding Z, Hu D, et al. Central role of lactic acidosis
in cancer cell resistance to glucose deprivation-induced cell
death. J Pathol 2012; 227: 189–99. doi: 10.1002/path.3978.
3. Fiaschi T, Marini A, Giannoni E, et al. Reciprocal
metabolic reprogramming through lactate shuttle coordi-
nately influences tumor-stroma interplay. Cancer Res 2012;
72: 5130–40.
4. Solyanik GI, Fedorchuk AG, Pyaskovskaya ON, et al.
Anticancer activity of aconitine-containing herbal extract BC1.
Exp Oncol 2004; 26: 307–11.
5. Pyaskovskaya ON. Antiangiogenic action of cyclophos-
phamide to experimental metastatic tumors. J Med Chem
2012; 2: 25–9 (in Ukrainian).
6. Kolesnik DL, Pyaskovskaya ON, Tregubova NV,
Solyanik GI. Lewis lung carcinoma variant with a high sen-
sitivity to antitumor antiangiogenic therapy exhibits a high
capacity for autophagy. Cytol Genet 2012; 46: 155–60.
doi: 10.3103/S009545271203005X.
7. Stacpoole PW. The pharmacology of dichloroacetate.
Metabolism 1989; 38: 1124–44.
8. Bonnet S, Archer SL, Allalunis-Turner J, et al. A mi-
tochondria-K+ channel axis is suppressed in cancer and its
normalization promotes apoptosis and inhibits cancer growth.
Cancer Cell 2007; 11: 37–51.
9. Wong JY, Huggins GS, Debidda M, et al. Dichloroace-
tate induces apoptosis in endometrial cancer cells. Gynecol
Oncol 2008; 109: 394–402. doi: 10.1016/j.ygyno.2008.01.038.
10. Michelakis ED, Sutendra G, Dromparis P, et al. Meta-
bolic modulation of glioblastoma with dichloroacetate. Sci
Transl Med 2010; 2: 31–4. doi: 10.1126/scitranslmed.3000677.
11. Stockwin LH, Yu SX, Borgel S, et al. Sodium dichloro-
acetate selectively targets cells with defects in the mitochondrial
ETC Int J Cancer 2010; 127; 2510–19.
12. Kumar A, Kant S, Singh SM. Novel molecular
mechanisms of antitumor action of dichloroacetate against
T cell lymphoma: Implication of altered glucose metabolism,
pH homeostasis and cell survival regulation. Chem Biol In-
teract 2012; 199: 29–37.
13. Pyaskovskaya ON, Dasyukevich OI, Kolesnik DL,
et al. Changes in VEGF level and tumor growth characteristics
during Lewis lung carcinoma progression towards cis-DDP
resistance. Exp Oncol 2007; 29: 197–202.
14. Biochemical methods (lipid and energy metabolism).
MI Prohorova, ed. L.: Leningrad Univ, 1982. 272 p.
15. Wang H, Joseph JA. Quantifying cellular oxidative
stress by dichlorofluorescein assay using microplate reader.
Free Radic Biol Med 1999; 27: 612–6.
16. Pyaskovskaya ON, Sorokina LV, Kolesnik DL, et al.
Dynamics of changes of antioxidant system indexes during
the growth of two Lewis lung carcinoma variants. Exp Oncol
2014; 36: 29–33.
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