Impact of lactic acidosis on the survival of lewis lung carcinoma cells
Aim: To investigate the effect of lactic acidosis on the survival of Lewis lung carcinoma cells under glucose-deprived conditions. Materials and Methods: LLC/R9 variant of Lewis lung carcinoma cells was cultured in glucose deficit or complete culture medium. Conditions of lactic acidosis, lactosis,...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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| Цитувати: | Impact of lactic acidosis on the survival of lewis lung carcinoma cells / D.L. Kolesnik, O.N. Pyaskovskaya, G.I. Solyanik // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 112–116. — Бібліогр.: 29 назв. — англ. |
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irk-123456789-1379732018-06-18T03:12:12Z Impact of lactic acidosis on the survival of lewis lung carcinoma cells Kolesnik, D.L. Pyaskovskaya, O.N. Solyanik, G.I. Original contributions Aim: To investigate the effect of lactic acidosis on the survival of Lewis lung carcinoma cells under glucose-deprived conditions. Materials and Methods: LLC/R9 variant of Lewis lung carcinoma cells was cultured in glucose deficit or complete culture medium. Conditions of lactic acidosis, lactosis, and acidosis were generated in glucose deficit medium. Cell survival, cell cycle, apoptosis, autophagy, and the content of glucose, lactate, vascular endothelial growth factor in the culture medium were determined. Light and fluorescent microscopy, flow cytometry, spectrophotometry, and ELISA were used. Results: It has been found that 24 h incubation of tumor cells under lactic acidosis caused (i) the reduction of the number of living cells by 33% (p < 0.05) and 56% (p < 0.05); (ii) the inhibition of apoptosis by 4.3-fold (p < 0.05) and 3.3-fold (p < 0.05); (iii) the reduction of the rate of glucose consumption by 2-fold (p < 0.05) and 2.5-fold (p < 0.05); (iv) an increase of lactate production more than twice (p < 0.05) and 1.6-fold (p < 0.05) compared with these indexes under conditions of glucose deficiency or complete glucose-containing medium, respectively. However, on the second day of culture under lactic acidosis, the number of viable cells reached a maximum, in contrast to culture in the complete medium. The number of live cells on the seventh day of culture under lactic acidosis exceeded almost 2–3 times (p < 0.05) that in the culture under conditions of the glucose deprivation or in complete medium. On the third day under lactic acidosis the autophagolysosomes count was 54% (p < 0.05) lower that that under glucose deficit. Conclusions: Lactic acidosis promoted the survival and proliferation of Lewis lung carcinoma cells by energy system reprogramming directed on inhibition of apoptosis and autophagy, a significant decrease in the rate of glucose utilization and activation of glutaminolysis and, consequently, increase of the lactate production rate. Inhibition of lactate production by tumor cells may be considered as a promising approach for more efficient antiangiogenic treatment of cancer. 2017 Article Impact of lactic acidosis on the survival of lewis lung carcinoma cells / D.L. Kolesnik, O.N. Pyaskovskaya, G.I. Solyanik // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 112–116. — Бібліогр.: 29 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137973 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Kolesnik, D.L. Pyaskovskaya, O.N. Solyanik, G.I. Impact of lactic acidosis on the survival of lewis lung carcinoma cells Experimental Oncology |
| description |
Aim: To investigate the effect of lactic acidosis on the survival of Lewis lung carcinoma cells under glucose-deprived conditions. Materials and Methods: LLC/R9 variant of Lewis lung carcinoma cells was cultured in glucose deficit or complete culture medium. Conditions of lactic acidosis, lactosis, and acidosis were generated in glucose deficit medium. Cell survival, cell cycle, apoptosis, autophagy, and the content of glucose, lactate, vascular endothelial growth factor in the culture medium were determined. Light and fluorescent microscopy, flow cytometry, spectrophotometry, and ELISA were used. Results: It has been found that 24 h incubation of tumor cells under lactic acidosis caused (i) the reduction of the number of living cells by 33% (p < 0.05) and 56% (p < 0.05); (ii) the inhibition of apoptosis by 4.3-fold (p < 0.05) and 3.3-fold (p < 0.05); (iii) the reduction of the rate of glucose consumption by 2-fold (p < 0.05) and 2.5-fold (p < 0.05); (iv) an increase of lactate production more than twice (p < 0.05) and 1.6-fold (p < 0.05) compared with these indexes under conditions of glucose deficiency or complete glucose-containing medium, respectively. However, on the second day of culture under lactic acidosis, the number of viable cells reached a maximum, in contrast to culture in the complete medium. The number of live cells on the seventh day of culture under lactic acidosis exceeded almost 2–3 times (p < 0.05) that in the culture under conditions of the glucose deprivation or in complete medium. On the third day under lactic acidosis the autophagolysosomes count was 54% (p < 0.05) lower that that under glucose deficit. Conclusions: Lactic acidosis promoted the survival and proliferation of Lewis lung carcinoma cells by energy system reprogramming directed on inhibition of apoptosis and autophagy, a significant decrease in the rate of glucose utilization and activation of glutaminolysis and, consequently, increase of the lactate production rate. Inhibition of lactate production by tumor cells may be considered as a promising approach for more efficient antiangiogenic treatment of cancer. |
| format |
Article |
| author |
Kolesnik, D.L. Pyaskovskaya, O.N. Solyanik, G.I. |
| author_facet |
Kolesnik, D.L. Pyaskovskaya, O.N. Solyanik, G.I. |
| author_sort |
Kolesnik, D.L. |
| title |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| title_short |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| title_full |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| title_fullStr |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| title_full_unstemmed |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| title_sort |
impact of lactic acidosis on the survival of lewis lung carcinoma cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2017 |
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Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/137973 |
| citation_txt |
Impact of lactic acidosis on the survival of lewis lung carcinoma cells / D.L. Kolesnik, O.N. Pyaskovskaya, G.I. Solyanik // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 112–116. — Бібліогр.: 29 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T04:51:26Z |
| last_indexed |
2025-07-10T04:51:26Z |
| _version_ |
1837234220817186816 |
| fulltext |
112 Experimental Oncology 39, 112–116, 2017 (June)
IMPACT OF LACTIC ACIDOSIS ON THE SURVIVAL
OF LEWIS LUNG CARCINOMA CELLS
D.L. Kolesnik*, O.N. Pyaskovskaya, G.I. Solyanik
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: To investigate the effect of lactic acidosis on the survival of Lewis lung carcinoma cells under glucose-deprived conditions. Materials
and Methods: LLC/R9 variant of Lewis lung carcinoma cells was cultured in glucose deficit or complete culture medium. Conditions
of lactic acidosis, lactosis, and acidosis were generated in glucose deficit medium. Cell survival, cell cycle, apoptosis, autophagy,
and the content of glucose, lactate, vascular endothelial growth factor in the culture medium were determined. Light and fluorescent
microscopy, flow cytometry, spectrophotometry, and ELISA were used. Results: It has been found that 24 h incubation of tumor cells
under lactic acidosis caused (i) the reduction of the number of living cells by 33% (p < 0.05) and 56% (p < 0.05); (ii) the inhibition
of apoptosis by 4.3-fold (p < 0.05) and 3.3-fold (p < 0.05); (iii) the reduction of the rate of glucose consumption by 2-fold (p < 0.05)
and 2.5-fold (p < 0.05); (iv) an increase of lactate production more than twice (p < 0.05) and 1.6-fold (p < 0.05) compared with these
indexes under conditions of glucose deficiency or complete glucose-containing medium, respectively. However, on the second day
of culture under lactic acidosis, the number of viable cells reached a maximum, in contrast to culture in the complete medium. The
number of live cells on the seventh day of culture under lactic acidosis exceeded almost 2–3 times (p < 0.05) that in the culture under
conditions of the glucose deprivation or in complete medium. On the third day under lactic acidosis the autophagolysosomes count was
54% (p < 0.05) lower that that under glucose deficit. Conclusions: Lactic acidosis promoted the survival and proliferation of Lewis lung
carcinoma cells by energy system reprogramming directed on inhibition of apoptosis and autophagy, a significant decrease in the rate
of glucose utilization and activation of glutaminolysis and, consequently, increase of the lactate production rate. Inhibition of lactate
production by tumor cells may be considered as a promising approach for more efficient antiangiogenic treatment of cancer.
Key Words: Lewis lung carcinoma, lactic acidosis, lactosis, glucose deficiency.
It is known that unlike normal tissue, microenvi-
ronment of malignant cells in a tumor characte rized
by a shortage of energy and the accumulation of plas-
tic substrates and their metabolic products, gradually
increasing with the distance from the vessel. This mi-
croenvironment occurs, in part, as a result of disability
of tumor angiogenesis, and causes the metabolic stress
in a tumor cell — the dominance of energy consump-
tion vs its production. Tumor cells cannot effectively
adapt to this metabolic stress because the predominant
mechanism of adenosine triphosphate (ATP) generation
in tumor cells is inefficient glycolysis. This offers the
prospect of inducing the death of cancer cells, creat-
ing or increasing imbalance between the requirement
of cells in energy and low level of its production (creating
a “metabolic catastrophe”) [1, 2].
Modern antiangiogenic cancer therapy is aimed
at inducing a “metabolic catastrophe” in tumor cells
by inhibiting tumor angiogenesis [3]. The efficiency
of antiangiogenic therapy depends on the simultane-
ous fulfillment of two conditions: effective inhibition
of tumor angiogenesis and tumor cell death induced
by nutrient deprivation, which is caused by a sig-
nificant reduction in tumor vascularity. Meanwhile,
in many cases, despite the effective inhibition of the
growth of blood vessels, expected antitumor action
doesn’t occur [4–7]. The reason for this lack of anti-
tumor effect is the tolerance of tumor cells to meta-
bolic stress. So, understanding the mechanisms that
ensure the survival of tumor cells under metabolic
stress is important for improving the efficiency of cur-
rent antiangiogenic cancer therapy and development
of new anticancer drugs.
The aim of the work was to investigate the effect
of lactic acidosis on the survival of Lewis lung car-
cinoma cells under conditions of metabolic stress
induced by glucose deprivation.
MATERIALS AND METHODS
Cell culture. In the work, LLC/R9 variant of Lewis
lung carcinoma cells was used [8]. Tumor cells are main-
tained in vitro in complete RPMI 1640 medium (Sigma,
USA) supplemented with 10% fetal calf serum (fetal
bovine serum (FBS), Sigma, USA) and 40 µg/ml gen-
tamicin at 37 °C in humidified atmosphere of 5% CO2.
To assess the survival of tumor cells under
lactic acidosis LLC/R9 cells at exponential growth
phase were seeded in 24-well plates (1.5 • 105 cells/
well) in complete RPMI 1640 medium and incubated
overnight. Then this medium was replaced with fresh
media, which differed by the content of glucose, lac-
tate, and pH (Table 1).
Table 1. Glucose and lactate content and pH of the culture medium
Culture conditions Glucose, mМ Lactate, mМ рН
Lactic acidosis 3.0 ± 0.1 14.0 ± 0.7 6.70 ± 0.01
Lactosis 3.0 ± 0.1 14.0 ± 0.7 7.40 ± 0.01
Acidosis 3.0 ± 0.1 1.6 ± 0.7 6.70 ± 0.01
Glucose deficit 3.0 ± 0.1 1.6 ± 0.1 7.40 ± 0.01
Complete medium 9.0 ± 0.5 1.6 ± 0.1 7.40 ± 0.01
For this, we used as a base glucose-free RPMI
1640 medium (Sigma, USA), which, after adding 10%
Submitted: May 06, 2017.
*Correspondence: E-mail: denkolesnik83@gmail.com
Abbreviations used: ATP — adenosine triphosphate; FBS — fetal
bovine serum; MDC — monodansylcadaverine; PBS — phosphate
buffered saline; VEGF — vascular endothelial growth factor.
Exp Oncol 2017
39, 2, 112–116
Experimental Oncology 39, 112–116, 2017 (June)39, 112–116, 2017 (June) (June) 113
FBS, was contained glucose and lactate in a final
concentration of 3.0 mM and 1.6 mM, respectively,
pH 7.4. Lactic acidosis was generated by adding lactic
acid (Sigma, USA) to the culture medium to a final con-
centration of 14.0 mM and pH 6.7; lactosis was gene-
rated by adding sodium lactate (Sigma, USA) to a final
concentration of 14.0 mM and pH 7.4; acidosis was
generated by adding HCl to a final pH of 6.7. Complete
culture medium after adding 10% of FBS contained
9.0 mM glucose, 1.6 mM lactate, pH 7.4. After replac-
ing the medium the cells were additionally incubated
under standard conditions for 7 days and then following
indexes were assessed: the number of living cells and
their cell cycle distribution, apoptosis rate, the level
of vascular endothelial growth factor (VEGF) produc-
tion, autophagy induction, glucose and lactate content
in the culture medium.
The number of cells and their viability was evalu-
ated by the routine method by direct counting using
a 0.4% solution of trypan blue.
Cell cycle distribution was assessed by flow cy-
tometry according to [9]. Briefly, the cells were resus-
pended in hypotonic lysis buffer (0.1% sodium citrate,
0.1% Triton X-100), which contained 5 mg/ml propi-
dium iodide, and DNA content was analyzed by flow
cytometry at a wavelength of 488 nm.
The number of apoptotic cells was determined
by staining with Hoechst 33258 dye (Sigma, USA) us-
ing a fluorescent microscope. To do this, the cells were
stained with Hoechst 33258 at a final concentration
of 1 mg/ml at 37 °C for 20 min, washed with saline and
then the changes in nuclear morphology were evalu-
ated, by counting at least 500 cells/sample. Apoptotic
cells were determined by the presence or fragmented
nuclei or condensed chromatin, apoptosis rate was
expressed as a percentage of total cells.
The VEGF level in cell culture medium was detected
by ELISA using mouse VEGF kit (Invitrogen, USA) accord-
ing to the manufacturer’s protocol. The rate of VEGF pro-
duction by tumor cells was calculated based on changes
in VEGF concentration in the culture medium and in the
number of living cells in the time course.
The level of autophagy in tumor cells was de-
termined using a fluorescent marker monodansylca-
daverine (MDC) (Sigma, USA) [10]. Shortly, the cells
were incubated with MDC at a final concentration
of 0.05 mM at 37 °C for 20 min, washed with phosphate
buffered saline (PBS) and lysed using 10 mM Tris-HCl
solution with the addition of 1% Triton X-100. Intracellu-
lar MDC levels were determined using fluorometer (ex-
citation filter 360/40 nm, emission filter 528/20 nm).
Fluorescence intensity was normalized to the number
of living cells.
Glucose level was measured in the medium
samples deproteinized with zinc hydroxide by the
enzymatic glucose-oxidative method using a set for
determining glucose in biological fluids (Sigma, USA)
according to the manufacturer’s protocol.
The level of lactate was measured in medium
samples deproteinized with perchloric acid by the
enzymatic spectrophotometric method using lactate
dehydrogenase (Sigma, USA) [11].
In all cases, the studied medium samples were
collected and stored at –20 °C prior to measurement.
The rate of glucose consumption and lactate pro-
duction in tumor cells was calculated based on the
concentration of substances in the culture medium and
changes in the number of living cells in the time course.
Statistical analysis. All measurements were done
in triplicate and data are presented as mean � s.e. Sta-� s.e. Sta- s.e. Sta-
tistical analysis was performed by descriptive methods
and Student’s t-test using Microsoft Excel software
and Microcal Origin (MicroSoft Inc., USA). p values
of less than 0.05 were considered as significant.
RESULTS
As it has been found lactic acidosis with glucose de-
privation significantly promoted survival of LLC/R9 cells.
Despite the slowing proliferation of the studied cells
on the 1st day of culture under lactic acidosis, already
on the 2nd day, their proliferation rates increased dra-
matically (Fig. 1). On the 1st day of culture under lactic
acidosis, the total number of living cells was almost
by 33% (p < 0.05), 41% (p < 0.05), and 56% (p < 0.05)
lower than that under conditions of the deficit of glu-
cose, lactose, and complete medium, respectively.
At the same time the maximal number of living cells,
which was achieved under lactic acidosis on the 2nd day,
almost 1.5-fold (p < 0.05) exceeded that under condi-
tions of the glucose deprivation, lactosis and acidosis.
In addition, the maximal number of tumor cells achieved
under lactic acidosis with glucose depletion did not
differ significantly from the corresponding value under
conditions of cell incubation in complete medium.
In the remote terms, the number of cells that sur-
vived on the 7th day of incubation (without replacement
of the media) under conditions of lactic acidosis and aci-
dosis (in both cases at pH 6.7) was almost by 2–3-fold
(p < 0.05) higher than that in conditions of glucose de-
privation or complete medium (in both cases, at pH 7.4).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 1 2 3 4 5 6 7
Ce
ll
co
un
t (
·1
06 )
Time (day)
Glucose deficit Acidosis Lactosis
Lactic acidosis Complete medium
Fig. 1. Growth kinetics of LLC/R9 cells in vitro under different
culture conditions
On the 1st day of cell culture, lactic acidosis caused
the reduction of glucose consumption rate by cells,
which clearly correlated with a decrease in prolifera-
114 Experimental Oncology 39, 112–116, 2017 (June)
tion rate at this time. In the conditions of lactic acidosis
this index was almost twice lower (p < 0.05) compared
to that under glucose and lactose deficiency and
2.5-fold lower (p < 0.05) than that in the complete
medium (Table 2). Acidosis, as well as lactic acidosis,
led to a significant reduction of the mentioned index
to 0.200 � 0.003 µmol/(106 cells•h) (p < 0.05), which
was associated with the lowest proliferation rate of tu-
mor cells and the presence of glucose in culture medium
even on the 4th day, unlike all other conditions of incuba-
tion, and the highest cell survival even on the 7th day.
Interestingly, along with the low rate of glucose
consumption by cells under lactic acidosis their lactate
production rate was the highest (p < 0.05) compared
to all other culture conditions (see Table 2). Moreover,
the ratio between lactate production and glucose con-
sumption significantly exceeded an expected value
in the case of an effective glycolysis.
Table 2. Glucose consumption and lactate production rates by LLC/
R9 cells under different culture conditions
Culture conditions Glucose consumption rate,
μmol/(106 cells•h)
Lactate production rate,
μmol/(106 cells•h)
Glucose deficit 0.328 ± 0.001 0.646 ± 0.003
Acidosis 0.197 ± 0.003 0.278 ± 0.022
Lactosis 0.319 ± 0.003 0.746 ± 0.204
Lactic acidosis 0.164 ± 0.012 1.328 ± 0.048
Complete medium 0.417 ± 0.030 0.807 ± 0.055
Lactic acidosis significantly reduced the percent-
age of apoptotic cells (Fig. 2). So, if at the 1st day
apoptosis rate was almost the same in all cases, start-
ing from the 2nd day the percentage of apoptotic cells
under lactic acidosis was 4.3-fold (p < 0.05), 5-fold
(p < 0.05) and 3.3-fold (p < 0.05) lower compared
to that under conditions of glucose deprivation, lac-
tosis and complete medium, respectively.
0
5
10
15
20
25
30
1 2 3
Ap
op
to
tic
c
el
ls
(%
)
Incubation time (day)
Glucose deficit
*
* *
Acidosis
Lactosis
Lactic acidosis
Complete medium
Fig. 2. Apoptosis level under different culture conditions.
*p < 0.05 as compared to that in the conditions of glucose deficiency
Significant changes in the cell cycle distribution related
to the culture conditions were not observed (Table 3).
Hence, inhibition of cell proliferation, found in the condi-
tions of lactic acidosis on the 1st day and in conditions
of acidosis during 1–3 days apparently occurred due
to blocking cell proliferation in all phases of the cell cycle.
There have been no significant effect of lactic aci-
dosis at the background of glucose deficiency on VEGF
production in LLC/R9 cells (Fig. 3). At least, during the
1st day, VEGF production rate in cells LLC/R9 in condi-
tions of lactic acidosis did not differ significantly from
that of all other culture conditions.
Table 3. LLC/R9 cell cycle distribution under different culture conditions
Culture
conditions Phase Incubation time, day
1 2 4 7
Lactic aci-
dosis
G0/G1 53.5 ± 1.0 61.6 ± 1.2 60.9 ± 0.5 44.8 ± 0.3
S 36.4 ± 0.1 27.0 ± 1.1 33.7 ± 1.2 48.8 ± 0.6
G2/M 10.1 ± 1.0 11.4 ± 0.1 5.5 ± 0.7 6.5 ± 0.8
Acidosis G0/G1 58.4 ± 0.2 62.3 ± 2.2 55.5 ± 0.8 41.8 ± 0
S 31.1 ± 0.6 25.5 ± 1.5 37.4 ± 0.7 45.2 ± 1.0
G2/M 10.5 ± 0.4 12.2 ± 0.7 7.2 ± 0.1 13.0 ± 1.0
Glucose
deficit
G0/G1 53.9 ± 0.7 59.8 ± 0.8 58.0 ± 0.7 47.6 ± 0.2
S 35.4 ± 1.0 23.6 ± 0.2 34.2 ± 0.9 45.9 ± 1.2
G2/M 10.7 ± 0.2 16.6 ± 1.0 7.8 ± 0.2 6.5 ± 1.1
Lactosis G0/G1 55.1 ± 1.5 61.5 ± 0.4 65.2 ± 0.6 52.6 ± 1.6
S 34.6 ± 1.8 23.1 ± 0.6 27.1 ± 0.2 39.1 ± 0.7
G2/M 10.3 ± 0.2 15.4 ± 0.1 7.7 ± 0.3 8.4 ± 1.0
Complete
medium
G0/G1 59.0 ± 0.9 57.8 ± 0.7 66.1 ± 1.3 56.5 ± 3.3
S 30.7 ± 0.3 26.0 ± 0.1 28.8 ± 1.5 36.2 ± 1.7
G2/M 10.3 ± 0.5 16.2 ± 0.8 4.7 ± 0.3 7.3 ± 1.6
0
10
20
30
40
50
Glucose deficit
Acidosis
Lactosis
Lactic acidosis
Complete medium
VE
GF
p
ro
du
ct
io
n
(p
g/
(1
06 c
el
ls
·h
))
Fig. 3. VEGF production by LLC/R9 cells under different culture
conditions
Induction of autophagy in tumor cells was tested
using MDC, which is considered as an autopha-
golysosomal marker due to its ability to accumulate
in cytoplasmic vacuoles. The data on the number
of lysosomes in LLC/R9 cells are shown in Fig. 4.
On the 3rd day of culture, maximal induction of tumor
cell autophagy under glucose depletion was ob-
served in the conditions of the glucose deficiency
and lactosis. Instead, autophagy induction under aci-
dosis and lactic acidosis was almost 49% (p < 0.05)
and 54% (p < 0.05) lower than that in the conditions
of glucose deprivation.
Glucose
deficit
Lactic
acidosis
Complete
medium
Acidosis Lactosis
M
DC
s
ta
in
in
g
(r.
u.
/(1
06 c
el
ls
)
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
* * *
Fig. 4. The number of lysosomes in LLC/R9 cells under different
culture conditions. *p < 0.05 as compared to that in the condi- compared to that in the condi-compared to that in the condi- to that in the condi-to that in the condi- that in the condi-that in the condi- in the condi-in the condi-
tions of deficiency of glucose and lactosis
DISCUSSION
It is known that the microenvironment of tumor cells
is usually characterized by the deficiency of nutrient
substrates and accumulation of metabolic products,
including excessive lactate and consequently low
extracellular pH [12]. Despite metabolic stress gene-
rated by malignant cells and their high dependence
on energy and nutrient substrates, they show tolerance
Experimental Oncology 39, 112–116, 2017 (June)39, 112–116, 2017 (June) (June) 115
to adverse conditions and provide not only survival but
also highly proliferative and metastatic activity.
One of the mechanisms of malignant cells survival
in adverse conditions, including nutrient deficiency, is limit-
ing cell proliferation and/or transition into dormancy [13].
The mechanisms of survival of tumor cells also include
a process of autophagy, i.e. lysosomal degradation
of intracellular components providing malignant cells with
an alternative source of nutrients and energy substrates
[13–16]. Growth factors, including VEGF, induce tumor
angiogenesis that results in the enhancement of supply-
ing tumor with oxygen and nutrients [17]. The resistance
of tumor cells to metabolic stress can also be associated
with impaired mechanisms of apoptotic death [18]. Repro-
gramming of energy metabolism, for example, increased
glucose uptake and activation of glycolysis enzymes
on the background of hypoxia, is one of the key mecha-
nisms of survival of malignant cells, as they almost always
exist in unfavorable metabolic microenvironment [19].
In this work lactic acidosis with glucose depriva-
tion significantly promoted cell survival of metastatic
Lewis lung carcinoma LLC/R9 cells. However, to sur-
vive in such conditions, tumor cells at the initial terms
of their incubation significantly inhibited the processes
requiring major energy consumption, such as prolifera-
tion, apoptosis, and autophagy.
Thus, when the cells just began to adapt to the con-
ditions of lactic acidosis the significant slowing of pro-
liferation has been observed. In the same period lactic
acidosis caused a significant inhibition of apoptosis and
autophagy, whose level under these conditions in the
LLC/R9 cells was the lowest among all other culture con-
ditions. Obviously, in terms of further enhancement of nu-
trient substrates deficiency on the background of lactic
acidosis one could expect induction of autophagy pro-
cesses for cell survival. At least in the later periods of cell
incubation under conditions of unfed culture, in these
cells there was observed activation of autophagy [20].
Slowing of the proliferation of LLC/R9 cells, ob-
served in the early stages of incubation under lactic
acidosis, was accompanied by a significant decrease
in the rate of glucose utilization by these cells. If in the
complete culture medium the rate of glucose con-
sumption was the highest, then in the conditions
of lactic acidosis it was the lowest. This index under
lactic acidosis was almost twice lower than that in the
conditions of glucose deprivation and lactosis.
Low consumption of glucose by tumor cells led to the
fact that glucose was determined in the culture medium
even on the 3rd day under lactic acidosis, while in the
conditions of glucose deficiency and lactosis the media
were completely depleted by glucose at the 2nd day.
Interestingly, the lowest rate of glucose consumption
in conditions of lactic acidosis was accompanied with the
highest rate of lactate production by tumor cells. More-
over, the level of lactate in the culture medium of these
cells is much higher than this, expected in the case
of complete metabolism of glucose through glycolysis.
These changes evidenced on a significant adjustment
in the regulation of energy metabolism of LLC/R9 cells,
caused by lactic acidosis. These changes can be ex-
plained by activation of oxidative phosphorylation and
glutaminolysis under conditions of lactic acidosis. The
latter was confirmed by the high speed of lactate produc-
tion in LLC/R9 cells and the high levels of lactate in the
culture medium under lactic acidosis. One should note that
lactosis, unlike lactic acidosis, did not lead to such a pro-
nounced increase in lactate production rate in LLC/R9 cells
and consecutive activation of glutaminolysis.
As it is known, glutaminolysis is a major (after gly-
colysis) way to provide rapidly tumor cells with energy.
Activation of glutaminolysis in tumor cells by extracellu-
lar lactate was shown in the work [21], in which signaling
cascades that are activated with lactate were thoroughly
investigated. It was found that in oxygenated malignant
human SiHa and HeLa cells lactate enters the cell
through monocarboxylate transporter 1 (MCT1), which
stabilizes HIF-2α, which transactivates c-Myc, which
in turn enhances the absorption of glutamine by cells
and activates oxidative phosphorylation.
The important role of lactate in the survival of tumor
cells is also confirmed by its ability to induce tumor
angiogenesis through the activation of HIF-1α [22]
as well as the ability of malignant cells and normal cells
co-opted in the tumor process, to use it as an energy
substrate [23, 24]. We also know that lactate can cause
degradation of the extracellular matrix, increase tumor
cell migration activity, which is known to correlate with
their metastatic potential [25, 26].
Therefore, the main adaptive reactions aimed at the
conservation of mentioned ATP-consuming processes
in LLC/R9 cells and related to their subsequent survival
under lactic acidosis occurred only during the 1st day
of culture. These reactions included dramatic inhibition
of proliferation, apoptosis, and autophagy, decreased
glucose consumption rate and activation of glutami-
nolysis. Starting from the 2nd day the active proliferation
of these cells resumed: the maximal number of live cells
under lactic acidosis was as high as that in complete
medium. The levels of dead and apoptotic cells under
lactic acidosis were the lowest not only in terms of the
initial incubation, but also for the whole culture duration.
Interestingly, acidosis, as well as lactic acidosis con-
tributed to increased survival of LLC/R9 cells. Obviously,
under acidosis, these cells used similar mechanisms
for their survival. However, slowing of the proliferation
of these cells under acidosis was much more pro-
nounced than that in conditions of lactic acidosis, and
continued for up to 3 days of culture. In addition, lactate
production in LLC/R9 cells under acidosis was signifi-
cantly lower than that in the conditions of lactic acidosis.
However, exactly acidosis provided the maximal survival
of LLC/R9 cells on the 7th day in all the studied culture
conditions. Thus, the increased survival of tumor cells
under acidosis was associated with a slowing of prolife-
ration and inhibition of glycolysis.
The ability of acidosis to promote survival
of LLC/R9 cells was also confirmed by the data
on their survival in extreme conditions of incubation,
namely in PBS supplemented with 10% FBS. As shown
116 Experimental Oncology 39, 112–116, 2017 (June)
in Table 4, survival of LLC/R9 cells after 3-day incuba-
tion in PBS, pH 6.7 was twice higher (p < 0.05), than
that in the conditions of incubation in PBS, pH 7.4.
Table 4. Survival of LLC/R9 cells under incubation in PBS
with different рН
Culture condition Viable cell count (106 cells)
0 day 3rd day
Control
(PBS + 10% FBS, рН 7.4) 0.15 ± 0.02 0.0319 ± 0.003
Acidosis
(PBS + 10% FBS, рН 6.7) 0.15 ± 0.02 0.0763 ± 0.009*
Note: *p < 0.05 as compared to control.
The role of acidification of the microenvironment
of tumor cells, but rather an important contribution
to the excess of hydrogen ions in the extracellular
environment to their survival is discussed in several
studies [27–29]. This feature of the tumor microenviron-
ment could be related, in particular, to a predominance
of cells with glycolytic phenotype and is actively con-
sidered as a promising target for anticancer therapy.
Thus, our findings suggested that acidosis pro-
moted survival of tumor cells, but unlike lactic acidosis
provided lower proliferative potential. Lactic acidosis not
only contributed to the survival of tumor cells but also
allow malignant cells actively proliferate under glucose
deprivation. Activation of proliferation of LLC/R9 cell was
preceded by a short period of adaptation to metabolic
stress, which includes reprogramming of its energy
system, aimed at energy conservation by inhibiting
apoptosis and autophagy, a significant decrease in the
rate of glucose utilization and activation of glutaminoly-
sis and as a result, increased lactate production rate.
Inhibition of lactate production by tumor cells may
be viewed as a promising approach for improving the
efficiency of antiangiogenic cancer therapy.
REFERENCES
1. Jin S, DiPaola RS, Mathew R, White E. Metabolic
catastrophe as a means to cancer cell death. J Cell Sci 2007;
120 (Pt 3): 379–83.
2. Teicher BA, Linehan WM, Helman LJ. Targeting cancer
metabolism. Clin Cancer Res 2012; 18: 5537–45.
3. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic
target. Nature 2005; 438: 967–74.
4. Ebos JM, Lee CR, Cruz-Munoz W, et al. Accellerated
metastasis after shorth-term treatment with a potent inhibitor
of tumor angiogenesis. Cancer Cell 2009; 15: 232–9.
5. Pàez-Ribes M, Allen E, Hudock J, et al. Antiangiogenic
therapy elicits malignant progression of tumors to increased
local invasion and distant metastasis. Cancer Cell 2009;
15: 220–31.
6. Rapisarda A, Melillo G. Role of the hypoxic tumor mi-
croenvironment in the resistance to anti-angiogenic therapies.
Drug Resist Updat 2009; 12: 74–80.
7. Loges S, Mazzone M, Hohensinner P, Carmeliet P. Si-
lencing or fueling metastasis with VEGF inhibitors: antiangio-
genesis revisited. Cancer Cell 2009; 15: 167–70.
8. 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.
9. Nicoletti I, Migliorati G, Pagliacci MC, et al. A rapid
and simple method for measuring thymocyte apoptosis
by propidium iodide staining and flow cytometry. J Immunol
Methods 1991; 139: 271–9.
10. Biederbick A, Kern HF, Elsässer HP. Monodansyl-
cadaverine (MDC) is a specific in vivo marker for autophagic
vacuoles. Eur J Cell Biol 1995; 66: 3–14.
11. Biochemical methods (lipid and energy metabolism).
Prohorova MI, ed. Leningrad: Leningrad Univ, 1982. 272 p.
(in Russian).
12. Calorini L, Peppicelli S, Bianchini F. Extracellular
acidity as favouring factor of tumor progression and metastatic
dissemination. Exp Oncol 2012; 34: 79–84.
13. Wu H, Ding Z, Hu D, et al. Central role of lactic aci-
dosis in cancer cell resistance to glucose deprivation-induced
cell death. J Pathol 2012; 227: 189–99.
14. Levine B, Klionsky DJ. Development by self-
digestion: molecular mechanisms and biological functions
of autophagy. Dev Cell 2004; 6: 463–77.
15. Sato K, Tsuchihara K, Fujii S, et al. Autophagy is acti-
vated in colorectal cancer cells and contributes to the tolerance
to nutrient deprivation. Cancer Res 2007; 67: 9677–84.
16. Casado P, Bilanges B, Rajeeve V, et al. Environmental
stress affects the activity of metabolic and growth factor signal-
ing networks and induces autophagy markers in MCF7 breast
cancer cells. Mol Cell Proteomics 2014; 13: 836–48.
17. Maj E, Papiernik D, Wietrzyk J. Antiangiogenic cancer
treatment: the great discovery and greater complexity (Review).
Int J Oncol 2016; 49: 1773–84.
18. Hanahan D, Weinberg RA. Hallmarks of cancer: the
next generation. Cell 2011; 144: 646–74.
19. Diaz-Ruiz R, Rigoulet M, Devin A. The Warburg and
Crabtree effects: On the origin of cancer cell energy metabolism
and of yeast glucose repression. Biochim Biophys Acta 2011;
1807: 568–76.
20. Kolesnik DL, Pyaskovskaya ON, Tregubova NA, Soly-
anik GI. Lewis lung carcinoma variant with a high sensitivity
to antitumor antiangiogenic therapy exhibits a high capacity for
autophagy. Tsitol Genet 2012; 46 (3): 33–40 (in Ukrainian).
21. Pérez-Escuredo J, Dadhich RK, Dhup S, et al. Lactate
promotes glutamine uptake and metabolism in oxidative cancer
cells. Cell Cycle 2016; 15: 72–83.
22. De Saedeleer CJ, Copetti T, Porporato PE, et al. Lac-
tate activates HIF-1 in oxidative but not in Warburg-phenotype
human tumor cells. PLoS One 2012; 7: e46571.
23. Sonveaux P, Végran F, Schroeder T, et al. Targeting
lactate-fueled respiration selectively kills hypoxic tumor cells
in mice. J Clin Invest 2008; 118: 3930–42.
24. Rattigan YI, Patel BB, Ackerstaff E, et al. Lactate
is a mediator of metabolic cooperation between stromal car-
cinoma associated fibroblasts and glycolytic tumor cells in the
tumor microenvironment. Exp Cell Res 2012; 318: 326–35.
25. Gatenby RA, Gillies RJ. Why do cancers have high
aerobic glycolysis? Nat Rev Cancer 2004; 4: 891–9.
26. Kennedy KM, Dewhirst MW. Tumor metabolism
of lactate: the influence and therapeutic potential for MCT
and CD 147 regulation. Future Oncol 2010; 6: 127–48.
27. McCarty MF, Whitaker J. Manipulating tumor acidi-
fication as a cancer treatment strategy. Altern Med Rev 2010;
15: 264–72.
28. Koltai T. Cancer: fundamentals behind pH targeting
and the double-edged approach. Onco Targets Ther 2016;
9: 6343–60.
29. Hu X, Chao M, Wu H. Central role of lactate and
proton in cancer cell resistance to glucose deprivation and
its clinical translation. Signal Transduct Targeted Ther 2017;
2: e16047.
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