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Glycolysis Inhibition and its Effect of Doxorubicin-Resistance in Neuroblas-toma
Jonathan F. Bean, Yi-Yong Qui, Songtau Yu, Sandra Clark, Fei Chu,Mary Beth Madonna
PII: S0022-3468(14)00046-3DOI: doi: 10.1016/j.jpedsurg.2014.01.037Reference: YJPSU 56668
To appear in: Journal of Pediatric Surgery
Received date: 24 January 2014Accepted date: 27 January 2014
Please cite this article as: Bean Jonathan F., Qui Yi-Yong, Yu Songtau, ClarkSandra, Chu Fei, Madonna Mary Beth, Glycolysis Inhibition and its Effect ofDoxorubicin-Resistance in Neuroblastoma, Journal of Pediatric Surgery (2014), doi:10.1016/j.jpedsurg.2014.01.037
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Glycolysis Inhibition and its Effect of Doxorubicin-Resistance in Neuroblastoma
Jonathan F. Bean1,2
, Yi-Yong Qui2, Songtau Yu
2, Sandra Clark
2,
Fei Chu2, Mary Beth Madonna
1,2
Institutions:
1. Department of Pediatric Surgery, Ann & Robert H. Lurie Children’s Hospital of
Chicago
2. Cancer Biology and Epigenetics, Children’s Hospital of Chicago Research Center
Corresponding Authors: Fei Chu, Mary Beth Madonna
Mailing Address:
Ann & Robert H. Lurie Children's Hospital of Chicago
Department of Pediatric Surgery
225 E. Chicago Avenue, Box 63
Chicago, IL 60611
Email Addresses: [email protected]; [email protected]
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Abstract
Background/Purpose: A common trait among cancers is the increased level of glycolysis
despite adequate oxygen levels to support aerobic respiration. This has been shown
repeatedly in different human malignancies. Glycolysis inhibitors, especially 3-
bromopyruvate, have been shown to be effective chemotherapeutic agents. The effect of
glycolysis inhibition upon chemotherapy resistance is relatively unknown.
Methods: Wild-type and doxorubicin-resistant lines of neuroblastoma (SK-N-SH and SK-
N-Be(2)C) were used in this study. Using an MTT assay, the IC50 of 3-BrPA was
determined. Subsequently, doxorubicin-resistant cell lines were treated with 3-
bromopyruvate, doxorubicin, and 3-bromopyruvate with doxorubicin. Additionally, a
luminescence ATP detection assay was used to measure intracellular ATP levels, and a
lactate assay was used to determine intracellular lactate levels. All experiments were
repeated in hypoxic conditions.
Results: Treatment with 3-bromopyruvate and doxorubicin significantly decreased the
mean cell viabilities at 24, 48, and 72 hours in normoxic conditions. A similar response
was replicated in hypoxic conditions. Treatment with 3-bromopyruvate significantly
decreased intracellular ATP and lactate levels.
Conclusion: Glycolysis inhibitors such as 3-bromopyruvate could prove to become an
effective means by which chemotherapy resistance can be overcome in human
neuroblastoma.
Keywords: glycolysis inhibition; neuroblastoma; ATP; 3-bromopyruvate; doxorubicin;
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Introduction
Early in the 20th
century, Warburg showed that a common trait of cancer cells is
the increased metabolic demand and increased utilization of glycolysis for energy
production.[Warburg 1956] This has been shown to be true in cancer cells that are
incubated in ambient oxygen levels ample to support aerobic respiration. As opposed to
non-malignant cells, cancers cells utilize glucose more rapidly, they have increased
production of lactate, and they have decreased demands for oxygen.[Bustamante 1977,
Wolf 2011] This inherent difference between malignant and non-malignant cells offers an
approach to treat cancer that would allow specificity of drug treatment against malignant
cells while sparing the non-malignant cells.
3-Bromopyruvate (3-BrPA), an inhibitor of hexokinase II and thus an inhibitor of
glycolysis, has been shown to be a potent chemotherapeutic agent. [Bhardway 2010,
Geschwind 2002, Levy 2012, Liu 2009, Munoz-Pineda 2003, Pereira 2009, Xu/Pelicano
2005] Many studies have shown the efficacy of 3-BrPA against various tumors,
including: breast, colon, hepatocellular, lymphoma, neuroblastoma, pancreatic, among
other cancers. Furthermore, 3-BrPA has been shown to effective as a monotherapy
against chemotherapy-resistant tumors.
Despite many studies showing the efficacy of glycolytic inhibitors against and
array of cancer cells, there have been relatively few studies examining the effect on
chemotherapeutic-resistant cancers of glycolysis inhibition in conjunction with standard
chemotherapeutic regimens on cancer cell response. One study showed that 3-BrPA
chemopotentiated the effects of platinum-based chemotherapeutics on colon and breast
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cancer cells.[Strandburg 2008] Another study demonstrated that 3-BrPA had a synergistic
effect with rapamycin in treatment of lymphoma and leukemic cells.[Xu/Pelicano 2005]
To date, there have been no studies examining the effects of 3-BrPA on the anti-cancer
effects of doxorubicin (Dox) on Dox-resistant human neuroblastoma.
The purpose of this study was to examine the effects of 3-BrPA upon Dox-resistant
human neuroblastoma cells as well as the effects of 3-BrPA on the response of Dox-
resistant tumor cells to treatment with Dox.
Methods
Human neuroblastoma (SK-N-SH and SK-N-Be(2)C) cell lines were purchased
from American Type Culture Collection (Rockville, MA). Medium and fetal bovine
serum were obtained from Mediatech (Herndon, VA) and Atlanta Biologicals (Atlanta,
GA), respectively. Doxorubicin (Dox), 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl
tetrazolium bromide (MTT), and 3-bromopyruvic acid (3-BrPA) were purchased from
Sigma (St. Louis, MO).
Initially, the IC50 of 3-BrPA was determined for wild-type and Dox-resistant
strains of both SK-N-SH and SK-N-Be(2)C using the MTT (2-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazoliumbromide) assay. Cells for each cell line were seeded into 96-
well plates (1x103 cells/well) and 3-BrPA was applied in a logarithmic manner from
10-7
M to 10-3
M. Untreated control groups were created for comparison. After 96 hours,
each well of the 96-well plate was treated with MTT (10 µL of 5 mg/ml solution) and
incubated for 4 hours at 37°C. Then 100 µL of 10% SDS/0.01 mmol/L HCL was added
to each well of the 96-well plate in order to solubilize the cells. The plate was then
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incubated for 15 hours at 37°C. The colorimetric absorbance of each well was determined
using an activation wavelength of 570 nm and a reference wavelength of 650 nm. Percent
cell viability for each treatment group was compared to the untreated control wells.
Next, cells were plated into four groups (Control, 3-BrPA treatment, Dox
treatment, and 3-BrPA+Dox treatment) using 6-well plates (6x104 cells/well). Each plate
was incubated at 37°C for 24, 48, and 72 hours in normoxic, ambient oxygen, conditions.
After each incubation period, cell viability for each treatment group was determined
using trypan blue staining and cell counting using a hemocytometer.
Subsequently, ATP levels were determined using the ATPLite Luminescence
ATP Assay kit (Perkin Elmer, Waltham, MA). Using white opaque, 96-well CulturPlates
(PerkinElmer, Waltham, MA), cells were plated into each well (1x103 cells/well). Four
treatment groups were created using equivalent concentrations of 3-BrPA, Dox, and 3-
BrPA+Dox as in the 6-well plates. An untreated control group was used for comparison.
After 48 hours of incubation at 37°C in normoxic, ambient oxygen, conditions, the ATP
levels were determined for each treatment group. Initially, 50μL of the provided
mammalian cell lysis solution was added to each well of the microplate and the plates
were mixed on an orbital shaker for 5 minutes to lyse the cells and stabilized the ATP.
50μL of the provided Luciferase/Luciferin substrate solution was applied to each well of
the microplate and mixed on the orbital shaker for 5 minutes. The plate was dark adapted
for 10 minutes and luminescence of each well was measured using a microplate reader to
determine relative ATP levels of each treatment group.
Intracellular lactate levels were determined using a lactate assay kit (Sigma-
Aldrich, St. Louis, MO). In the same manner as the cell viability studies, we plated the
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neuroblastoma cells into 6-well plates and re-created the four treatment groups (Control,
3-BrPA treatment, Dox treatment, and 3-BrPA+Dox treatment). After 48 hours of
incubation at 37°C in normoxic, ambient oxygen, conditions, the intracellular lactate
levels were determined for each treatment group and compared to controls.
Cell viability, ATP level, and intracellular lactate studies were repeated in
hypoxic conditions. This was achieved by incubating the cell culture plates at 37°C in a
modular hypoxia incubator chamber (Billups-Rothenberg, Del Mar, CA) filled with 89%
N2, 10% CO2, 1% O2 gas. The cell viability in hypoxia for SK-N-SH was measured for
each drug treatment group at 12, 24, and 36 hours of incubation in hypoxia; the ATP
level for each drug treatment group was measured after 12 hours of incubation in
hypoxia.
Each experiment was completed three times with differing passages of each cell
line to create experimental replicates.
Results
The IC50 of 3-BrPA for wild-type and Dox-resistant SK-N-SH as well as for SK-
N-Be(2)C was determined by MTT assay. The IC50 of 3-BrPA for SK-N-SH was 3.1x10-
5M and for SK-N-Be(2)C the IC50 was 7.6x10
-6M.
After determination of the IC50 of each cell line, the sub-IC50 treatment
concentrations of drugs for the SK-N-SH cells was chosen to be 2x10-5
M for 3-BrPA and
1x10-5
M for Dox and for SK-N-Be(2)C the drug concentrations of 3-BrPA and Dox were
5x10-6
M and 5x10-5
M, respectively.
The Dox-resistant cells were plated into 6-well culture plates. Four groups were
created including a 3-BrPA treatment group, a Dox treatment group, a 3-BrPA+Dox Fig 1; Table 1
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treatment group, and finally an untreated control group. These groups were incubated at
normoxic conditions for 24, 48, and 72 hours and then using trypan blue staining the cell
viability was determined.
Figure 1 and Table 1 show significant decreases in the mean cell viabilities for
those cells treated with 3-BrPA+Dox compared to those cells treated with either 3-BrPA
or Dox alone. At 24, 48, and 72 hours, 3-BrPA synergizes with Dox to decrease the
overall mean cell viability of those neuroblastoma cells treated with either drug
independently.
We repeated these experiments in hypoxic conditions. As stated previously, for
the SK-N-SH cells, it was necessary to shorten the incubation period due to the increased
susceptibility of that cell line to the effects of hypoxia. As can been seen in Table 1, the
synergistic effects of 3-BrPA in combination with Dox was preserved in hypoxia.
We replicated the 3-BrPA treatment group, Dox treatment group, the 3-
BrPA+Dox treatment group and the control using 96-well white opaque culture plates for
both SK-N-SH and SK-N-Be(2)C for the ATP studies. The results are shown in Table 2.
We saw that treatment with 3-BrPA consistently decreased the total amount of ATP in
both normoxia as well as hypoxia.
Finally, the intracellular lactate studies revealed that treatment with 3-BrPA
decreases lactate levels compared to controls. (Figure 2) This decrease in lactate levels
was significant in normoxic conditions.
Discussion
In this study of the effects of 3-BrPA on Dox-resistance in human neuroblastoma,
we have successfully shown that use of glycolytic inhibitors can effectively overcome the
Table 2
Fig 2
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chemotherapy resistance mechanisms in this cancer and improve its response to treatment
with doxorubicin. Treatment of Dox-resistant neuroblastoma cells with 3-BrPA
significantly improved the response of these cells to treatment with Dox over treatment
with either 3-BrPA or Dox alone. Furthermore, our evidence shows that this effect is
present in both normoxic as well as hypoxic conditions.
Prior studies of the use of glycolytic inhibitors such as 3-BrPA have repeatedly
shown the efficacy of glycolytic inhibitors in the treatment of various malignancies; but
prior to this study, few have focused on the effects of glycolysis inhibition on
chemotherapy resistance. In the 1970’s, multidrug resistance was shown to be mediated
through a transmembrane glycoprotein named P-glycoprotein. [Endicott 1989]
Subsequently, further studies revealed that P-glycoprotein is a member of the ATP-
binding cassette family, functions as an ATPase, and needs ATP in order to function.
[Shapiro 1994] This transmembrane protein actively transports chemotherapeutic drugs
from the intracellular space to the extracellular space conferring chemotherapy resistance.
Chemotherapy resistance in neuroblastoma is mediated through the MDR1 gene and P-
glycoprotein. Since various studies have shown that 3-BrPA decreases the levels of
intracellular ATP, it would be reasonable to assume that these drugs would also have an
effect on chemotherapy resistance. [Geschwind 2004, Ko 2001]
Through our study, we revealed that indeed treatment of Dox-resistant
neuroblastoma cells with 3-BrPA improved the response of those cells to Dox. As tumors
of any cancer often have areas of differing oxygen tensions, it was logical to test if this
effect was also present in hypoxic conditions. We were able to show through incubating
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these cancer cells in hypoxic conditions that the effect of 3-BrPA persisted despite
decreased ambient oxygen.
Through the use of the luminescence ATP detection system, we were able to see
that 3-BrPA decreased the total amount of intracellular ATP. Interestingly, the total
amount of ATP in the SK-N-SH control cell lines were greater that those of the SK-N-
Be(2)C cell lines despite plating equivalent numbers of cells in each ATP study. This
difference is possibly due to a higher baseline metabolic rate in the SK-N-SH cells and
would also account for the increased susceptibility of that line to hypoxia thus
necessitating decreased incubation intervals in the hypoxic chamber.
Finally, the lactate assay revealed that intracellular lactate levels were decreased
in our neuroblastoma lines when glycolysis was inhibited with 3-BrPA. Of note, the
lactate levels between the 3-BrPA treatment groups and the 3-BrPA+Dox treatment
groups showed no difference compared to the controls in the hypoxic environment. We
suggest that this reflects the overall baseline upregulation of lactate production of
neuroblastoma when exposed to a hypoxic environment.
Conclusion
In conclusion, we have found that in human neuroblastoma there is equal efficacy
of 3-BrPA against wild-type and Dox-resistant cell lines. Furthermore, we showed that 3-
BrPA seems to enhance the response of Dox-resistant neuroblastoma to treatment with
Dox and this enhancement exists in both normoxic conditions as well as hypoxic
conditions. Finally, our study suggests that this increased response of Dox-resistant cells
to treatment with Dox is mediated through decreased levels of glycolysis. Further work is
needed to more fully investigate the mechanism of action of 3-BrPA in the enhancement
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of chemotherapeutic susceptibility. This future work could prove to be the basis of a
unique mode of overcoming chemotherapy resistance in neuroblastoma.
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enhancement of death-inducing signaling complex formation and apical Procaspase-8
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Figure and Table Legends. (See attached files for the .jpeg images)
Figure 1. Cell viability at 24, 48, and 72 hours of incubation of SK-N-SH and SK-N-
Be(2)C Dox-resistant cells with 3-BrPA, Dox, or 3-BrPA+Dox compared to controls.
Table 1. Mean cell viabilities at 24, 48, and 72 hours for each experimental treatment
group as well as p-values.
Table 2. Relative ATP Levels
Figure 2. Intracellular lactate levels of SK-N-SH in normoxia and hypoxia.
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Figure 1
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Figure 2
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Table 1. IC50 values for SK-N-SH and SK-N-Be(2)C
SK-N-SH Wild-Type (95% CI) DoxR-6 (95% CI)
2.3x10-5M (1.9x10-5M to 3.0x10-5M) 3.1x10-5M (2.4x10-5M to 4.1x10-5M) SK-N-Be(2)C
Wild-Type (95% CI) DoxR-5 (95% CI) 4.6x10-6M (2.7x10-6M to 7.5x10-6M) 7.6x10-6M (3.9x10-6M to 1.5x10-5M)
Table 2. Percentage Mean cell viabilities at 24, 48, and 72 hours for each experimental treatment group as well as corresponding p-values
SK-N-SH DoxR-6 (Normoxia)
Control 3-BrPA Dox 3-
BrPA+Dox p-value (Dox vrs 3-
BrPA+Dox) 24
hours 87.03 ±
1.53 39.92 ±
2.57 41.23 ±
3.54 11.35 ±
1.69 p < 0.0001
48 hours
71.95 ± 2.47
34.03 ± 5.00
32.95 ± 2.86
4.42 ± 1.64 p < 0.0001
72 hours
83.42 ± 1.36
55.12 ± 2.46
45.20 ± 2.70
8.40 ± 1.40 p < 0.0001
SK-N-Be(2)C DoxR-5 (Normoxia)
Control 3-BrPA Dox 3-
BrPA+Dox p-value (Dox vrs 3-
BrPA+Dox) 24
hours 80.44 ±
3.45 37.10 ±
2.41 32.31 ±
5.15 7.61 ± 1.54 p = 0.0001
48 hours
70.37 ± 2.28
27.58 ± 2.53
31.88 ± 3.94
5.97 ± 1.13 p < 0.0001
72 hours
77.68 ± 1.75
32.06 ± 2.25
34.18 ± 4.69
3.11 ± 1.03 p < 0.0001
SK-N-SH DoxR-6 (Hypoxia)
Control 3-BrPA Dox 3-
BrPA+Dox p-value (Dox vrs 3-
BrPA+Dox) 24
hours 53.10 ±
3.35 22.38 ±
4.12 25.87 ±
4.86 10.83 ±
1.99 p = 0.0169
48 hours
22.00 ± 1.58
10.42 ± 0.76
13.62 ± 1.70
4.43 ± 1.21 p = 0.0013
72 hours
1.70 ± 0.85 - - - -
SK-N-Be(2)C DoxR-5 (Hypoxia)
Control 3-BrPA Dox 3-
BrPA+Dox p-value (Dox vrs 3-
BrPA+Dox) 24
hours 89.80 ±
3.46 35.05 ±
4.00 26.20 ±
2.64 6.38 ± 1.31 p < 0.0001
48 hours
67.67 ± 4.33
32.75 ± 3.11
20.33 ± 1.95
5.73 ± 1.43 p = 0.0001
72 69.27 ± 26.20 ± 17.58 ± 3.78 ± 1.11 p < 0.0001 hours 5.32 2.56 0.99
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AAP Discussion
Glycolysis inhibition and its Effect On Doxorubicin-Resistance in Neuroblastoma –
Jonathan Frederick Bean, MD, Chicago, IL
Discussant: DR. Cynthia DOWNARD, Louisville, KY
One quick question that I have is the 3-bromopyruvate is that a viable
in vivo model or will it kill the animals?
Response: DR. BEAN: Yes, it is. It actually has been used in
hepatocellular carcinoma, and actually in rat models, and they
are starting to use them in human studies now.
Discussant: DR. Jed Nuchtern. Houston, TX: I just wanted to
congratulate you on an excellent study. This very nice paper is
probably the beginning of some consensus that is arising in the
field of oncology about the central role that the mitochondria
may actually play in chemotherapy resistance. There was some
very nice work presented at the COG meeting recently showing that
if the mitochondria are changed in some way in a resistant tumor,
it doesn't matter what chemotherapy you give, it's not going to
work, so this I think is also a very interesting fact and
interesting study to bring to our attention that just shows that
as we go forward we really have to think about the mitochondria,
sort of a neglected organelle if you will.
