This document summarizes a study that investigated the effect of sodium dichloroacetate (DCA) on melanoma cells. The study found that DCA induced apoptosis in melanoma cells in a dose-dependent manner, with higher concentrations of DCA inducing apoptosis more rapidly. Leptin provided a protective effect against DCA-induced apoptosis. The results suggest that targeting cancer cell metabolism by switching them from glycolysis to oxidative phosphorylation through DCA treatment could be a potential therapeutic approach for treating melanoma.

1. Conclusions
1. Melanoma cells are sensitive to DCA-induced apoptosis.
2. The higher the concentration of DCA that is used, the more rapidly apoptosis
occurs.
3. Leptin provides a protective effect against DCA-induced apoptosis.
Results
• A 150 mM DCA treatment results in a 60% decrease in cell viability for both
incubation periods.
• A 50 mM DCA treatment results in a 20% decrease in cell viability for both
incubation periods.
• A 10 mM DCA treatment results in a >80% cell viability within 48 hours.
• Leptin treatment results in a protective effect for 10 and 50mM DCA. 150mM
DCA treatment saw only a slight protective effect.
Future Studies
Lactate dehydrogenate A (LDH-A) is an is form of lactate dehydrogenate is a major molecular mediator of
enhanced glycolysis followed by lactic acid fermentation. Lactate dehydrogenases are involved in the
conversion of pyruvate to lactate. Over-expression of LDH-A is linked to tumor hypoxia, angiogenic factor
production, and increased cellular acidity. Rather than using DCA as a means for sensitizing cancer cells to
apoptosis, we could knockdown LDH-A expression in melanoma cells. The results should be similar to those
of a treatment with DCA. The knockdown of LDH-A should help promote the shuttling of pyruvate into the
mitochondria, which would sensitize the cancer cells to apoptosis.
Dichloroacetate Induces Apoptosis in Melanoma Cells
Maxwell Schwam, Amber Jackson, Jacob Morgan, Dr. Elizabeth Brandon, Ph.D.
Department of Biology, Mississippi College, Clinton, Mississippi
Abstract
Drug development in oncology has focused on targets essential for the survival of all
dividing cells within an organism. This leads to a narrow therapeutic window. Cancer’s
remarkable adaptability explains much of the poor performance of cancer drugs. Selective
induction of apoptosis in cancer, but not in normal cells remains the biggest challenge to
oncologists. Most cancers are characterized by anaerobic glycolysis. Often called the Warburg
effect, cancer cells favor this metabolic pathway even over oxidative phosphorylation, which is
far more effective at generating ATP compared with glycolysis. An evolutionary theory of
carcinogenesis identifies a metabolic shift from oxidative phosphorylation to glycolysis as a
critical and early adaptive mechanism of cancer cells against apoptosis and hypoxia. Two
explanations for this exist: 1) reduced oxygen levels in tumors make oxidative phosphorylation
difficult and 2) oxidative phosphorylation sensitizes cells to apoptotic stimuli, of which there
many in cancer cells.. To test the hypothesis that melanoma cells undergo this metabolic
transition, which if reversed, could stimulate apoptosis, we treated cells with sodium
dichloroacetate (DCA). This drug in affect switches cancer cells from favorable glycolysis back
to glucose oxidation in mitochondria, consequently increasing cancer cells’ sensitivity to
apoptosis. B16F10 mouse melanoma cells were treated with 10, 50, and 150 mM for 24 and 48
hours. This drug in affect switches cancer cells from favorable glycolysis back to glucose
oxidation in mitochondria. The mitochondria in human cells hold a reservoir of apoptotic factors.
Administering DCA corrects the metabolism in cancer cells and as a result increases cancer cells
sensitivity to normal apoptosis. In our experiment, we looked at the affect DCA in different
concentrations on melanoma cells in vitro. There was a dose dependent response to DCA, with
150mM DCA causing a significant drop in cell viability. These results suggest that turning on
oxidative phosphorylation in melanoma cells could be a useful therapeutic approach.
Background
The war on cancer since 1971 remains ongoing and only a few battles have been won. Drug
development in oncology has focused on targets essential for the survival of all dividing cells within
an organism. As a result, this leads to a narrow therapeutic window. Cancers’ remarkable variation
and adaptability explains the poor performance of cancer drugs. Selective induction of cell death
(apoptosis) in cancer but not in normal cells remains the biggest challenge to oncologists. Within all
living cells there are proximal biochemical pathways that are crucial for cell survival. Targeting more
distal pathways that integrate several proximal signals is one way to address the problem of
heterogeneity in proximal pathways. Cancer cells’ unique metabolism is an ideal example.
Most cancers are characterized by anerobic glycolysis. This simply means cancers use
glycolysis as the primary pathway for energy production in the cell despite the presence of oxygen
within the cell. Often called the Warburg effect, cancer cells favor this metabolic pathway even over
glucose oxidation, which is far more effective at generating ATP compared with glycolysis. In order
to keep up with the demands of the cell through this inefficient shift in metabolism to primarily
glycolysis, cancer cells up-regulate glucose receptors and significantly increase glucose uptake in an
attempt to decrease the energy deficit. As a result, this bio-energetic difference between cancer and
normal cells, might offer a very selective therapeutic target, since glycolysis is not seen in normal
tissues apart from skeletal muscle tissue. Enzymes involved in glycolysis have been found to be
regulators of apoptosis and gene transcription, suggesting that links between metabolic sensors, cell
death, and gene transcription are established directly through the enzymes that control metabolism. An
evolutionary theory of carcinogenesis identifies metabolism and glycolysis as a critical and early
adaptive mechanism of cancer cells against hypoxia that persists because it offers resistance to
apoptosis in cancer cells.
Hypoxia-inducible factor (HIF) is activated in cancer cells by hypoxic conditions. HIF signals
the cancer cell to upregulate glucose transporters and enzymes required for glycolysis. HIF induces
the expression of pyruvate dehydrogenase kinase (PDK), a gate-keeping enzyme that regulates the
flux of carbohydrates (pyruvate) into the mitochondria. In the presence of activated PDK, pyruvate
dehydrogenase (PDH) is inhibited, which limits the entry of pyruvate into the mitochondria where
glucose oxidation continues. Sodium dichloroacetate (DCA) is a drug that switches cancer cells from
favorable glycolysis back to glucose oxidation in mitochondria. Administering DCA locks PDH in the
active conformation by inhibiting PDK and allowing for glucose oxidation in the mitochondria to
resume. As a result, the cancer cells become more sensitized to apoptotic stimuli, which triggers the
release of apoptotic factors out of the mitochondria. From this observation and other research in this
area, it’s possible that looking at a cancer cell’s metabolism could be the new therapeutic window we
have been looking for.
Numerous health problems and complications have been linked to obesity. Obesity is becoming
increasingly common in our society. Studies have shown a correlation between melanoma and obesity.
As a result, skin cancers are being diagnosed more often. Increased leptin, an adipokine secreted from
adipose tissue, has been observed in obese subjects. Leptin increases proportionally to body mass
index. Little is known about the role of leptin in melanoma. A possible window for investigation is
the survival effects of leptin in melanoma.
Methods
We looked at the affect of different concentrations of sodium dichloroacetate (DCA) on
melanoma cells in vitro. We performed three cell death assays in which we recorded the rate of
melanoma cell death in control and experimental cells to determine the dose of DCA that caused
the most cell death. Twelve well plates were used to grow the B16F10 mouse melanoma cells. We
grew cells until confluency in RPMI medium containing 1% streptomycin, 1% penicillin, 5% fetal
bovine serum (FBS), and 1.5 g/L sodium bicarbonate. DCA (10mM, 50mM, and 150mM) was
added and then we incubated the cells for 24 and 48 hours. To observe the ratio of live to dead
cells, we washed the cells with phosphate buffered saline (PBS), trypsinized , and then resuspended
them. We stained the cells with Trypan blue and counted them using a hemacytometer. We
determined the optimum conditions for the assay: number of cells/well, DCA concentration, and
duration of treatment. In all three experiments, we plated 100,000 cells in 12 well plates. In the
third experiment, we added leptin at 0.1ug/mL to our cell death assay.
WT
Incubation Periods
Figure 1. Cells were treated and incubated for a 24 hours period with either 10, 50, or 150 mM
DCA. Trypsinized cells were stained with Trypan blue and the total number of cells were counted
in a hemocytometer. Triplicate wells were counted for each treatment and timepoint. Results are
reported as the percentage of live cells.
Figure 2. Cells were treated and incubated for 48 hours with 10, 50, 150mM DCA.
Trypsinized cells were stained with Trypan blue and the total number of cells were
counted in a hemocytometer. Three replicate wells were counted for each treatment.
Results are reported as the percentage of live cells.
Figure 3. Cells were treated with different concentrations of DCA along with 0.1ug/mL
leptin and incubated for 24 hours. Trypsinized cells were stained with Trypan blue and
the total number of cells were counted in a hemocytometer. Replicate wells were counted
for each treatment. Results are reported as the percentage of live cells.
Untreated Cells DCA Treated Cells
Acknowledgements
This work was supported by the Mississippi INBRE funded by grants
from the National Center for Research Resources (5P20RR016476-11)
and the National Institute of General Medical Sciences (8 P20
GM103476-11) from the National Institutes of Health.
References
1. ED Michelakis, The Metabolism of Cancer Cells. British Journal of Cancer (2008), 989-994.
2.ED Michelakis, L Webster, and JR Mackey, Dichloroacetate (DCA) as a potential metabolic-targeting
therapy for cancer. (2008), 99(7): 989-994.
3.Jason Y.Y. Yong, Gordon S. Huggins, Marcella Debidda, Nikhil C. Munshi, and Immaculata De Vivo,
Dichloroacetate induces Apoptosis in Endometrial Cancer Cells. Gynecol Oncol. (2008); 109(3): 394-402.
Figure 4. Representative images of control and DCA-treated cells. Images were obtained using
a Leica DMIR 3000B inverted microscope with a Q-imaging 12-bit CCD monochrome camera.
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