Cho, Liu i

AbstractThe Illinois Junior Academy of Science

CATEGORY Microbiology STATE REGION # __ 5

SCHOOL Illinois Math and Science Academy IJAS SCHOOL # 5048

CITY/ZIP Aurora 60506 SCHOOL PHONE (630) 907-5000

SPONSOR Ms. Marie Olszewski and Dr. Morris Kletzel

NAME OF SCIENTIST*Hyunjii (Justina) Cho GRADE 11

NAME OF SCIENTIST Jimmy Liu GRADE 11

* If this project is awarded a monetary prize, the check will be written in this scientist’s name, and it will be his/her responsibility to distribute the prize money equally among all participating scientists.

PROJECT TITLE: The Analysis of Cancer Cells Through Examination of Morphology, Gene Expression, and Effects of Dichloroacetate

Purpose: Cancer currently stands as one of the leading causes of death. Recent studies have shown that “stem cells are the source of at least some, and perhaps all, cancers.” (NYTIMES, 2006). With this fairly recent breakthrough in cancer research, we aimed to detect the differences in morphology, gene expression, and the response to DCA, if any, between normal and cancer stem cells.

Procedure: We removed the mononuclear cells or stem cells by creating a density gradient with Ficoll-Paque. Once we extracted the mononuclear cells, we made our cell cultures. To determine how much the cells proliferated, we performed a cell count; then, we checked the cell viability. Flow cytometry and occasional RT-PCR procedures were followed and several different concentrations of DCA diluted in RPMI were given to cell culture. Finally, we observed the differences between cancerous and normal cells. Conclusion: Our research has shown that the concentration of DCA has a negative correlation with the amount of viable cells, thus successfully eradicating cancer cells by promoting apoptosis. Further concurrent tests conducted with DCA involve non-cancerous cells; if DCA is shown to have no effect on normal cells, then cancer research will be transformed.

Cho, Liu ii

Safety SheetThe Illinois Junior Academy of Science

Directions: The student is asked to read this introduction carefully, fill out the bottom of this sheet, and sign it. The science teacher and/or advisor must sign in the indicated space.

SAFETY AND THE STUDENT: Experimentation or research may involve an element of risk or injury to the student and to others. Recognition of such hazards and provision for adequate control measures are joint responsibilities of the student and the sponsor. Some of the more common risks encountered in research are those of electrical shock, infection from pathogenic organisms, uncontrolled reactions of incompatible chemicals, eye injury from materials or procedures, and fire in apparatus or work area. Countering these hazards and others with suitable controls is an integral part of good scientific research.In the space below, list the principal hazards associated with your project, if any, and what specific measures you have used as safeguards. Be sure to read the entire section in the Policy and Procedure Manual of the Illinois Junior Academy of Science entitled "SAFETY GUIDELINES FOR EXPERIMENTATION" before completing this form.

Safety Hazards:Use of human bloodUse of Dichloroacetate

Safety Precautions:Universal Safety PrecautionsAlways wear a lab coat, gloves, and lab goggles.Since dichloroacetate is corrosive, it needs to be handled with care.We always work under the supervision of our mentor

SIGNED________________________________________________________________________Student Exhibitor(s)

SIGNED________________________________________________________________________Sponsor**As a sponsor, I assume all responsibilities related to this project.

Cho, Liu iii

Humans as Test Subjects EndorsementThe Illinois Junior Academy of Science

THESE RULES WILL BE STRICTLY ENFORCED FOR THE STATE SCIENCE EXPOSITION. NO REGION SHOULD SEND A PROJECT TO THE STATE EXPOSITION THAT DOES NOT MEET THESE REGULATIONS.Students and sponsors doing a human vertebrate project must complete this form. The signature of the student or students and the sponsor indicates that the project was done within these rules and regulations. Failure to comply with these rules will mean the disqualification of the project at the state level. This form must follow the safety sheet in the project paper.(1) Humans must not be subjected to treatments that are considered hazardous and that could result in undue stress, injury, or death to the subject.(2) No cultures involving humans (mouth, throat, skin, or otherwise) will be allowed. However, cultures obtained from reputable biological suppliers or research facilities are suitable for student use.(3) Quantities of food and non-alcoholic beverages are limited to normal serving amounts or less. Normal serving amounts must be substantiated with reliable documentation. This documentation must be attached to the Humans as Test Subjects Endorsement form. No project may use over-the-counter or prescription drugs or any other chemical agents in order to measure their effect on a person.(4) The only human blood that may be used is that which is either purchased or obtained from a blood bank, hospital, or laboratory. No blood may be drawn by any person or from any person specifically for a science project. This rule does not preclude a student making use of data collected from blood tests not made exclusively for a science project.(5) Projects that involve exercise and its effect on pulse, respiration rate, blood pressure, and so on are allowed provided the exercise is not carried to the extreme. Electrical stimulation is not permitted. A valid, normal physical examination must be on file for each test subject. Documentation of same must be attached to the Humans as Test Subjects Endorsement form.(6) Projects that involve learning, ESP, motivation, hearing, vision, and surveys require the Humans as Test Subjects form.In this space, briefly describe the use of humans in your project. Use the back of page if necessary.

The signatures of the student or students and sponsor below indicate that the project conforms to the above rules of the Illinois Junior Academy of Science.

_________________________________________ ________________________________(Sponsor) (Student)

_________________________________ _________________________________________(Date) (Student)This form MUST be displayed on the front of the exhibitor’s display board. It may be reduced to 4.25” x 5.5

The blood that is being used in our experiment comes from the Children’s Memorial Hospital. We also used K562, a cancer cell line, which has been cultured in the stem cell laboratory at the Children’s Memorial Hospital. Patient information is kept confidential.

Cho, Liu 1

The Analysis of Cancer Cells through Examination of Morphology, Gene Expression,

and Effects of Dichloroacetate

Hyunjii Cho and Jimmy Liu

Illinois Mathematics and Science Academy

Cho, Liu 2

Table of Contents

Abstract i

Safety Sheet ii

Endorsements iii

Title Page 1

Table of Contents 2

Acknowledgements 3

Purpose and Hypothesis 4

Review of Literature 5

Materials 10

Methods of Procedure 13

Results 17

Discussion 23

Conclusion 26

Reference 27

Cho, Liu 3

Acknowledgements

We would like to thank Ms. Marie Olszewski, Dr. Morris Kletzel, and Wei Huang

for their continual support, valuable advice, and expert guidance throughout our

experiment. Without their help, our project would not have been possible. Also, we

would like to thank Ms. Judith A. Scheppler for her dedication to the Student Inquiry and

Research program.

Cho, Liu 4

Purpose

Many scholarly journals are delving into the depths of stem cell research; experts

have reason to believe that it is through stem cells that the cure for cancer will eventually

be discovered. Past studies on cancer focused on shrinking tumors by destroying cancer

cells, yet disregarded the remaining stem cells that spurn more cancer cells. This

investigation focuses on elucidating the mechanisms through which cancer operates. We

will do utilize cell surface markers, compare gene expression, and examine the

morphology of the stem cells. The main focus of the experiment will be to test the

efficacy of a novel new cancer-target drug called dichloroacetate through various assays

and cultures of the human myeloid leukemia K562 cell line. We hope to provide a basis

for further investigation in the search for the cure to cancer.

Hypothesis

We hypothesized that DCA would have a positive effect in restoring normal

apoptosis, or programmed cell death, in cancer cells, and effectively prohibit cell

proliferation. We also hypothesized that DCA would leave normal cells unaffected.

Cho, Liu 5

Review of Literature

In today’s rapidly evolving scientific world, much advancement has been made in

the field of cancer research. Because cancer stands as one of the leading causes of death,

we thought it would be worthwhile to contribute to the elucidation of mechanisms of

cancer. This research covers a wide array of topics, including cancer cell lines, stem cells,

dichloroacetate, stem cell markers, flow-cytometry, and polymerase chain reactions

(PCR). Selected literature is both pertinent and applicable to the experiment.

Hematopoietic stem cells are isolated from the peripheral blood or bone marrow

and are capable of self-renewal and differentiation into specialized cells. Apoptosis,

programmed cell death, is necessary in order to prevent an excessive number of cells.

Normal apoptotic functions are not present in cancerous stem cells, allowing unlimited

cell regeneration. In healthy stem cell division, the cell regenerates itself and also creates

a progenitor cell. Although the progenitor cell cannot regenerate itself, it is capable of

dividing indefinitely into mature specialized cells (Lee & Herlyn, 2007). The progenitor

cell then matures and become a specialized cell.

Stem cells are characterized by both “self-renewal and their ability to produce

cells that differentiate” (Morrison & Kimble, 2006), called progenitor cells. Stem cells

divide using two strategies: Asymmetric and Symmetric stem-call divisions. In

asymmetric stem-cell division, stem cells divide to produce one identical daughter of

itself, and one daughter that is capable of differentiating. Through this process, stem cells

are unable to expand in number and thereby unable to produce stem-cell pools that are

needed for development and for cell regeneration after injury. Therefore, there must be

another method that stem cells utilize to maintain control of their numbers. “Symmetric

Cho, Liu 6

divisions are defined as the generation of daughter cells that are destined to acquire the

same fate.” (Morrison et al., 2006). Through symmetric division, stem cells are able to

divide into two stem cell daughter cells or two progenitor cells. Most stem cells are able

to divide both asymmetrically and symmetrically, and these two modes are controlled by

developmental and environmental signals. However, this balance is sometimes disrupted

and defective in disease states.

Source: University of Medicine and Dentistry at New Jersey (2007).

Figure 1. Division of stem cells displaying self-renewal and the progenitor cell.

Cell surface markers are used to determine what type of cell is in a culture. The

cell markers bind to specialized proteins on the surface of every cell in the body called

Cho, Liu 7

receptors. Fluorescent tags are attached to surface markers (Figure 2) and these

fluorescent tags are then detected via flow cytometry, a technique which picks up

fluorescent light and thus identifies the cell surface markers.

Source: Appendix E: Stem Cell Markers (2001).

Figure 2. Identifying Cell Surface Markers Using Fluorescent Tags.

RT-PCR (reverse transcription polymerase chain reaction) is a method that

determines the frequency of expression of a certain gene by amplification. First, a strand

of RNA is reverse transcribed into a cDNA (complementary strand of DNA). Once a

cDNA strand is created, the next step is to anneal the upstream and downstream primers.

Source: Dolan DNA Learning Center (2007).

Figure 3. An upstream and downstream primer selected to amplify the gene of interest; i.e. the sequence of nucleotides between the primers.

Cho, Liu 8

Next, the primers elongate, and the overlapping sequence of DNA is the sequence

that will be amplified as the number of cycles increases.

Source: Dolan DNA Learning Center (2007).

Figure 4. Cycle three of the polymerase chain reaction; the separate sequence of interest has been amplified twice.

According to Kanato, Hosen, and Yanagihara, WT1 expression is restricted to

hematopoietic stem cells or progenitor cells. The Wilm’s Tumor 1 gene (WT1) is over-

expressed in leukemia, thus explaining the proliferation of blood cells causing leukemia

(2005). In normal subjects, there is low or undetectable expression of the WT1 gene;

however, this gene is widely expressed in leukemic cell lines, as well as in the majority of

lymphoid and myeloid leukemias of childhood and adulthood (Hernandez-Caballero,

Mayani, & Montesinos, 2007). The K562 human leukemia cell line is strikingly similar to

stem cells. According to Young and Hwang-chen, K562 has characteristics of self-

renewal and pluripotency similar to those of stem cells (1981, p.7073). Comparable to

leukemic stem cells, K562 also expresses the WT1 gene (Hidehiko, Masato, & Etsuko,

2006).

The Effects of Dichloroacetate on Cancer Stem Cells

Cho, Liu 9

Studies involving DCA and cancerous cells have been conducted previously, yet

the compound’s effects on cancerous stem cells may prove to yield results that will aid in

the detection of differences between cancerous and healthy stem cells. Cancerous cells

are characterized by apoptosis resistance. Factors that promote the absence of apoptosis

include lowered expression of the K+ channel Kv1.5 and higher mitochondrial membrane

potential. DCA activates Kv channels in all cancer cells, but not normal cells. These

effects collectively restore apoptosis, which, in turn, should result in the death of cancer

cells and shrinkage of tumors (Bonnet, et al., 2007). In order to determine if DCA has the

same effects on undifferentiated cancerous stem cells as on differentiated cancerous

mature cells, we will conduct two experiments; each will have a control group (no DCA

applied), and several experimental groups (DCA applied in varying concentrations). We

will then perform a cell count and graph the growth over time. If DCA has the same

effects on stem cells as on mature cells (restore apoptosis in cancerous cells only, leaving

healthy cells unaffected), then the experimental group (with DCA) in the leukemic stem

cell experiment should experience a slower growth in cell number as time progresses. In

addition, the experimental group (with DCA) in the healthy stem cell experiment should

exhibit the same growth patterns as the control group, proving that DCA has no effect on

healthy stem cells ((Bonnet, et al., 2007). Does DCA have an innocuous effect on normal

cells and provide to be a promising solution to eradicating cancer cells?

Cho, Liu 10

Materials

Because multiple trials were conducted in order to ensure a large sample size and

therefore accurate results, the quantity of materials required is indefinite.

Isolating mononuclear cells by creating a density gradient

1L 1:2 Ratio, Ficoll-Paque to Blood

1L Phosphate-buffered Saline (PBS)

Test tubes

One (1) Centrifuge

Pipettes

Blood

Micropipettes (One of each: 1000μL, 100μL, 10μL)

Dichloroacetate (DCA)

1mL Dichloroacetate 99% concentration

1L RPMI tissue culture

Ten (10) 50mL large test tubes

Micropipettes (One of each: 1000μL, 100μL, 10μL)

Culturing Cells

1L RPMI Medium

Ten (10) Cell Plates with twenty-four (24) wells each

One (1) Incubator

Cho, Liu 11

5mL GMSF Cytokine

Pipettes

Cell Count

One (1) Cell-Dyn cell counter

Cells to count

Cell Viability

One (1) Hemacytometer

One (1) Microscope

10 mL Trypan Blue

One (1) Manual cell counter

Reverse Transcriptase Polymerase Chain Reaction

RNA Primers

Thermo-cycler

RNA Polymerase

Agarose Gel Electrophoresis

Flow Cytometry

Flow Cytometer

Reagents

Cho, Liu 12

Explanation of Materials and Apparatus.

Ficoll-Paque creates a density gradient.

Centrifuge is used to spin samples in test tubes in order to extract the accumulated pellet

of mononuclear cells.

GMSF Cytokine stimulates cell proliferation.

Cell Dyne cell counter is used by inserting the needle into the sample and pressing the

lever behind it.

Hemacytometer is used as a slide to view the cells treated with trypan blue under a

microscope for cell viability.

Trypan Blue permeates into dead cells; therefore it is used to discern dead and live cells.

Cho, Liu 13

Methods of Procedure

Isolating mononuclear cells by creating a density gradient

A. Take an initial cell count (Pre-dilution (purely blood))

a. Mix the sample well.

b. Put the needle into sample.

c. Initiate the Cell Dyne cell counter by pressing the pedal.

d. Wait for data

B. Create a density gradient

a. Add Ficoll-Paque to a test tube

i. Label the test tube.

ii. Avoid touching the Ficoll-Paque bottle in order to keep the sample

sterile.

b. Dilute the blood sample by adding a ratio of 1:1 of PBS:blood.

c. Mix well.

d. Add the diluted sample slowly to the Ficoll (1 Ficoll: 2 Blood)

e. Place the test tube in centrifuge

i. Balance with a similar weight test tube

ii. Run at 3000rpm for 10 minutes with no brake

f. Remove from the centrifuge. Separated cells should look like Figure 1.

Cho, Liu 14

Figure 1. Density gradient using Ficoll-paque.

C. Remove the mononuclear cells

a. Remove half of the plasma/PBS

b. Put the mononuclear cells into a new test tube

D. Wash cells

a. Fill the remainder of the test tube containing mononuclear cells with PBS

b. Mix well.

c. Place the test tube into centrifuge

d. Remove from centrifuge and pour out PBS/plasma. Cells should appear as

a pellet at the bottom (Figure 2).

Cho, Liu 15

Figure 2. Pellet at the bottom of a test tube formed from the accumulation of mononuclear cells.

E. Prepare to take a final cell count

a. Add 2mL of RPMI tissue culture to your pellet of cells.

b. Mix well to break up cell pellet.

c. Take cell count using the Cell Dyne machine.

Culturing Cells

Preparing to culture cells

A. Use bleach to disinfect the hood.

B. Calculate the amount of cells per well (1.0 x 10^6 cells)

C. Add RPMI to cells to fill cell well to 1mL

D. Pipette desired amount of cells to each well

E. Place in incubator

a. Add autoclaved deionized bottom tray of incubator

b. Add cupric sulfate to bottom tray of incubator

Cho, Liu 16

Cell Count

A. Obtain sample and operate the Cell Dyn 1700 machine

B. Place the needle of the Cell Dyn 1700 machine into the sample and press the lever

to activate the cell counter.

Cell Viability with Trypan Blue

A. Extract 10 µL of the cell sample and place it in small test tube

B. Add 20 µL of trypan blue to the cells and mix well

C. Extract 10 µL of mixture and pipette into hemocytometer, which should be

cleaned with alcohol pads

D. View the contents on the hemacytometer under a microscope.

E. Count the number of viable cells and the number of dead cells. (Dead cells are

permeated with trypan blue, while live cells contain no blue color and instead

emanate a glow)

DCA Concentration tests

A. Calculate the amount of DCA needed to make specific molar concentrations

B. Mix DCA with RPMI to make x Molar solution

C. Fill plate wells to 1mL.

Cho, Liu 17

Results

Figure 5 displays the increase in K562 cell concentration as time increases, thus

confirming the indefinite proliferation of K562. The initial cell concentrations of K562 at

D0 were 0.1 K/uL.

K562 Control (Jan. 16.08- Jan. 30. 08)

y = 0.0385e1.0877x

R2 = 0.9541

0

0.5

1

1.5

2

2.5

3

3.5

D0 D3 D10 D14

Time (days)

Co

nce

ntr

atio

n (

K/u

L)

Figure 5. Bar graph of the cell concentration as time increases. K562 was cultured and the cell concentration of the controls (without DCA) were recorded at various times in a 14 day period.

Cho, Liu 18

The graph in figure 6 displays a decrease in cell concentration of K562 as the DCA

concentration increases.

Figure 6. Protocol a: The effects of increasing DCA concentration on the cell concentration of K562. Cell concentration (K/uL) of K562 cell cultures with no DCA and cultures with 2.5 mM, 5mM, 10mM, and 20mM were observed after 168hrs after the addition of DCA.

The highest concetrations of DCA from protocol a were tested again in another

set of experiments, protocol b. The resulting K562 cell concentrations in 10 mM and

20mM DCA after 96 hours of the addition of DCA shows to be far less than that of the

control measured at the same time.

Cho, Liu 19

Figure 7. Protocol b: K562 cell concentrations on day 14 of the culture and 96 hours after adding DCA to certain cultures. 10mM and 20mM DCA concentrations were tested and compared to the day 14 control culture.

Protocol c consisted of BM2 normal bone marrow stem cell cultures at different

concentrations. The effects of the various DCA concentrations on BM2 were relatively

similar. At day 0 of the culture, the initial cell concentration was 2.0K/uL. At day 16 of

the culture and 48 hours after the addition of DCA to some of the wells of the culture, the

sample was observed. The concentrations were relatively around 1.7K/uL. There were no

significant differences between the BM2 cell concentration of the control and the DCA-

treated cultures.

Cho, Liu 20

Protocol c: BM2 March 7 Day 16 (48Hrs in DCA)

1.72 1.7 1.72 1.7333333

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Control 5mM 10mM 20mM

DCA Concentration (mM)

Ce

ll C

on

ce

ntr

ati

on

(K

/uL

)

Figure 8. Protocol c: BM2, a normal stem cell sample, was treated with various concentrations of DCA. The resulting cell concentrations after 48 hrs of immersion in DCA were compared to the control, which had been cultured for a total of 14 days.

A secondary result of experimentation is the analysis of gene expression. Again,

the most significant difference can be evidenced through a comparison of non-DCA

treated cultures and DCA-treated cultures. As shown in Figure 9, when a pristine culture

of K562 cells is used as the baseline, the two cultures treated with 20 mM of DCA from

both protocol a and b have low expressions of WT1, the cell proliferation gene. BM2, the

normal stem cell sample, also has a very low, almost undetectable, expression of WT1.

The control K562 samples and the 10mM DCA concentration K562 samples all express

the WT1 relatively higher than the 20mM DCA treated K562 samples.

Cho, Liu 21

Figure 9. WT1 expression in K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2. The resulting data was examined through the LightCycler Data Analysis.

Through flow cytometry, certain characteristics of the normal and cancer cells

were determined. In protocol a, 23.53% of the K562 control cells were double positive

for CD123 and CD 34. CD123 is a cell surface marker for cells capable of proliferation,

while CD34 is a marker for hematopoietic stem cells. The percentage of cells that are

both positive steadily increases as the concentration increases. Table 1 shows the results

of flow cytometry detecting the markers of CD 123 and CD34 on the control K562, K562

treated with 10mM DCA, and K562 treated with 20 mM DCA.

Tabe 1. The percentage of cells that are both CD123 and CD34. Flow cytometry was performed to determine the types of cell surface markers on the cells.

Protocol a CD123+ and CD34+K562 control 28.53%10mM DCA treated 49.86%20mM DCA treated 48.95%

Flow cytometry was also performed on normal stem cells and only 1.50% of the cells

were both CD123 and CD34 positive.

Cho, Liu 22

Cho, Liu 23

Discussion

The results of this experimentation provide a foundation on which to continue

furthering studies in oncology. This research covers gene expression, analysis of DCA,

and morphology. Because obtaining stem cells was difficult, much of this experiment was

based on the K562 cell line. The K562 leukemia cell exhibit the WT1 gene, similarly to

leukemia stem cells and also has “properties of self-renewal and pluripotency similar to

those of the hematopoietic stem cell” (Young & Hwang-Chen, 1981).

In the first set of experiments (protocol a), various concentrations of DCA were

tested on cultures of K562 that initially began at concentrations of 0.1K/uL: K562 control

group (n=2), K562 group with 2.5mM (n=2), 5mM DCA (n=2), K562 group with 10mM

DCA (n=2), and 20mM DCA. The control group, which was not treated with DCA,

continued normal cell proliferation. The DCA-treated groups exhibited a decreased

concentration of cells (Figure 6), indicating decreased cell proliferation. 10 mM and 20

mM DCA concentrations were chosen for the second set of experiments (protocol B) on

K562 because they exhibited almost no increase in cell concentration. The initial cell

concentration was 0.1 K/uL, while the cell concentrations of K562 cells in 10mM and

20mM DCA for 168hrs only had 0.2 K/uL cell concentrations. This is not a significant

increase and can be accounted for by few trials, and human and machine (Cell Dyn 1700)

error. Protocol b confirmed that DCA may inhibit cell proliferation because it also

showed that cell concentrations did not increase significantly from the initial cell

concentration of 0.1 K/uL and they also did not proliferate as much as the control did

(Figure 7).

Cho, Liu 24

According to figure 8, DCA had no significant affect on the cell concentrations of

BM2. This shows that while DCA affects cancerous cells, it does not affect normal cells.

More tests will need to be performed on normal cells in the future, in order to wholly

support our conclusion. Due to the lack of normal bone marrow stem cells available, we

were not able to perform many tests on normal stem cells.

RT-PCR was performed on protocol a and protocol b. The expression of WT1 in

protocol a and b was measured through RT-PCR. In protocol a, the K562 group treated

with 10mM of DCA showed that the cells still expressed the Wilm’s tumor 1 gene

(WT1), which regulates cell proliferation. However, the cells in protocol a that were

treated with 20mM of DCA did not significantly exhibit the WT1 gene. The test was

repeated for protocol b and similar results were achieved. This shows that as the

concentration of DCA increases, the proliferation of cancer cells was inhibited.

Our findings in the morphology through analysis of stem cell markers contradict

the discoveries in WT1 expression. Through flow cytometry, we found that the

percentage of K562 cells that are both CD123 and CD 34 positive increases as the

concentration of DCA increases. This means that the percentage of K562 cells that are

capable of proliferating have a positive correlation with DCA concentrations. The stem

cell marker CD123 may be present on cells for more than just capability of proliferation;

therefore, the RT-PCR results would prove to be more reliable. Gene expression is more

specific in determining function than the stem cell markers. In order to understand why

the two assays disagree, one would have to investigate whether other factors cause the

presence of CD123 on cells.

Cho, Liu 25

The prospect of dichloroacetate as a novel, therapeutic cancer treatment is

realistic because it restores mitochondrial function, one of the fundamental pathways

unique to the progression of cancer. Additionally, as it has been used for many decades in

the treatment of metabolic diseases, it is known to be relatively non-toxic (Bonnet, et al.,

2007). The most significant property of dichloroacetate is that it has no effect on normal

cells (Bonnet, et al., 2007).

Cho, Liu 26

Conclusion

Our results corroborate our hypothesis that dichloroacetate would inhibit cell

proliferation and are highly indicative of the veracity of DCA as a cancer-targeting drug.

The 20 mM DCA concentration seemed to be most effective. In protocol a, after 168 hrs

in 20mM DCA, K562 showed little to no proliferation. Protocol b showed similar results.

This, however, does little to show the affects of DCA on normal stem cells. Thus, we

tested 10 and 20mM concentrations of DCA on BM2 and found that the cell

concentrations did not significantly change. As evidenced in protocols a and b, these

results suggest that DCA decreases cell proliferation in cancer cells as its concentration

increase.

In the future, more tests on normal stem cells and cancer stem cells will need to

be performed. Since this is an ongoing project, we will aim to establish a more solid

foundation for the effectiveness of DCA by testing the DCA on a mixed culture of

normal and cancer stem cells.

Because it was difficult to obtain a sufficient amount of cancer and normal stem

cells, it was difficult to compare normal and cancerous stem cells. We were unable to do

multiple trials of the effect of DCA on normal stem cells. However, we determined that

using DCA as a future treatment for cancer is possible.

Cho, Liu 27

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