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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