The Effects of Vitamin C and D3 on A549 Cells

By

Angelica Bautista and Schyler Jones

A Senior Thesis Submitted in Partial Fulfillment

Of the Requirements for Graduation

Central Magnet School

April 2018

Thesis Committee:
Dr. Mary Farone
Mrs. Eve Harrison
Dr. Melanie Thomas
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS ii

ACKNOWLEDGEMENTS

We would like to first thank our Stem IV teacher, Mrs. Eve Harrison, for all of her

guidance in our thesis and supporting us with ideas and suggestions to improve our research.

Mrs. Harrison taught us about APA formatting, and she was always prepared to answer any of

our questions. Our thesis would not have been produced smoothly without her assistance.

We would also like to thank our English teacher, Dr. Thomas, for constantly reminding

us about deadlines and proofreading our paragraphs. Even though she had a large portion of the

senior class as her students, she was consistently open to answer any thesis question. We greatly

appreciate all her hard work and her everlasting support.

Finally, our thesis would not have been possible without the help of our patient,

hardworking mentor. Dr. Mary Farone, a Biology professor from Middle Tennessee State

University, edited our methodology greatly and provided almost all of our experiment’s

materials. She watched over us as we performed the experiment and kindly corrected our

mistakes. Dr. Farone was very willing to help us schedule our multiple lab meetings and to offer

her own expertise.

THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS……………………………………………………………………..ii

ABSTRACT……………………………………………………………………………...………iv

INTRODUCTION……………………………………………………..………………………1-6

METHODOLOGY………………………………………………………………….……..…7-12

Figure #1………………………………………………………………………...............10

RESULTS…………………………………………………………………………..………..13-14

Figure #2……………………………………………………………………………...…13

Figure #3…………………………………………………………………………….......14

Figure #4………………………………………………………………………………...14

DISCUSSION……….……………………………………………………………………….15-17

CONCLUSION……………………………………………………………………………...18-19

REFERENCES………………………………………………………..…………….……....20-21
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS iv

ABSTRACT

The purpose of this research is to test the effects of vitamin C and D3 on A549 lung
cancer cells to determine which vitamin treatment is more effective in reducing the cells’ area
and count. Based on previous research, vitamin C is known to break down into hydrogen
peroxide and cause damage to cells. Additionally, vitamin D3 is known to produce cyclin-
dependent kinases to suppress cancer cell expansion. Therefore, the treatment combining vitamin
C and D3 is reasonably expected to decrease cell size and count the most out of all treatments.
A549 lung cancer cells were given six treatments with only one of them combining vitamin C
and D3. Unexpectedly, the vitamin D3 treatment at 2 micromolars decreased the cells’ area and
count more than the combination treatment. These results are significant because they can
expand cancer research on vitamin therapy.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 1

INTRODUCTION

Research Question

How do varying combinations of Vitamin C and D3 affect cell viability and cell area of

A549 cells in cell cultures after being incubated for 48 hours?

Research Purpose

The purpose of this experiment is to examine the effects of Vitamin C and D3 on the

amount and size of A549 cells, which are non-small cell lung cancer cells. Studies have shown

that vitamin D3 reduces the growth of cancer cells and suppresses their cell cycle (Kennel &

Drake, 2013). Vitamin C reacts with iron produced by free radicals and cancer cells which

oxidatively damages the cell (Park, 2017). Since both vitamins individually impair cancer cells,

combining them together could reduce the A549 cell amount and area more efficiently. This

information could provide significant data for the research of current cancer treatments.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 2

BACKGROUND INFORMATION

Lung Cancer

Lung cancer is the second leading cause of cancer death in men and women alike,

encompassing 14% of all new cancers. In fact, one of every four cancer deaths is due to lung

cancer. Lung cancer survival depends on the stage, as people in an earlier cancer stage have a

higher rate of survival (“Key Statistics for Lung Cancer,” 2016). The three main types of lung

cancer are Non-Small Cell Lung Cancer, Small Cell Lung Cancer, and Lung Carcinoid Tumors.

Non-Small Cell Lung Cancer is the most common, accounting for about 85% of lung cancers.

Subtypes include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Small

Cell Lung Cancer is also known as oat cell cancer. It accounts for about 10-15% of lung cancers,

and it spreads very quickly. Lastly, Lung Carcinoid Tumors account for only 5% of lung cancers,

and they are also called neuroendocrine tumors, many of which grow slowly and do not tend to

spread (“Lung Cancer,” n.d.). The majority of lung cancers are preventable, as they are mainly

due to smoking, secondhand smoke, or radon exposure. However, there are lung cancers with an

unknown cause, for which preventability is not known (“Lung Cancer Prevention and Early

Detection,” n.d.). Diagnosis is done through laboratory tests, chest x-rays, CT scans, biopsies,

bronchoscopies, and light and electron microscopy. Non-Small Cell Lung Cancer has three

stages: limited, extensive, and recurrent. The treatments currently available are surgery,

chemotherapy, radiation therapy, laser therapy, and endoscopic stent placement (“Small Cell

Lung Cancer,” 2017). Stages of Non-Small Cell Lung Cancer are Stage 0, I, II, IIIA, IIIB, and

IV. Treatments for this type of cancer are determined by the stage of cancer and include those

previously mentioned for Small Cell Lung Cancer, as well as electrocautery, chemoprevention,

and radiosensitizers (“Non-Small Cell Lung Cancer Treatment,” 2017). However, these
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 3

treatments are not enough— 155,870 deaths occur each year due to lung cancer (Key Statistics

for Lung Cancer, 2016).

A549 Lung Cancer Cell Line

A cell line is a group of research cancer cells that keep dividing and growing under

certain conditions in a laboratory. There are over 200 lung cell lines, but the one that was used

for the experiment was the A549 lung cancer cell line, which has been widely studied. The A549

lung cancer cell line was taken from a 58-year-old Caucasian male (“A549 (ATCC® CCL-

185™),” n.d.). The cell line is a continuous tumor-cell line that was taken from a human lung

carcinoma in 1976. It is a Non-Small Cell Lung Cancer that has type II alveolar epithelial cell

properties (Gazdar, Girard, Lockwood, Lam, & Minna, 1993). The cell line has been used to

study the inhibition of cancer cell proliferation, the effects of insulin, and to culture A549 cells in

different media. It can synthesize lecithin utilizing the cytidine diphosphocholine pathway and

may have inclusion bodies (“A549 Cell Line human 86012804,” n.d.). Due to these facts, the

A549 cell line is a logical cell line to use for our experiment. However, the cells can be adherent,

so they will need to be trypsinized (“Removal of Adherent Cells from Culture Surface Using

Trypsin,” n.d.).

Vitamin C

Vitamin C, also known as ascorbic acid, is an essential nutrient that can be derived from

fruits and vegetables, such as oranges, strawberries, and kale. This vitamin is widely known to

help produce collagen, a protein in charge of the state of our bones, tendons, skin, blood vessels,

etc (Nordqvist, 2017). Simply consuming foods that contain vitamin C suppresses the organic

compound's ability to damage tumor cells because the chemicals in the digestive system
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 4

immediately break down the vitamin; it must be injected directly into the bloodstream to have its

full, anti-cancer effects. This organic compound has the ability to break down into hydrogen

peroxide, which damages body tissue and DNA; therefore, it has the ability to damage cancer

cells. Normal, healthy cells produce catalase, an enzyme that catalyzes the decomposition of

hydrogen peroxide to water and oxygen. On the other hand, tumor cells produce a low amount of

catalase; thus, they are more likely to be exposed and damaged by hydrogen peroxide produced

by vitamin C (Lerche, 2017).

Three Singaporean scientists, Raymond Yuen Chuen Fong, Glenda Chong Sze Ling, and

Meng Lim Kah, have conducted a case study on the effects of Intravenous Vitamin C (IVC)

therapy on cancer patients at the Hosanna Clinic in Singapore. Nine patients with different types

of cancer were chosen and given a certain amount (25-100g) of vitamin C dosage based on their

plasma vitamin C levels. As soon as they started the IVC therapy, the patients were encouraged

to strictly follow a Gerson diet consisting of low amounts of sugar, carbohydrates, and meat

along with high amounts of fruits and vegetables. In relation to lung cancer, patient three was a

54 year old Chinese woman with stage IV lung cancer. The patient was given a prognosis of

seven months left to live; however, after being put on IVC therapy, she lived past seven months.

The cancer in her lungs decreased in size due to a combination of IVC therapy and gefitinib,

which is a drug that targets non-small cell lung cancer. This case study concluded that “...for

most patients, IVC therapy has improved their quality of life and may have prolonged their life

expectancy” (Fong, Ling, & Kah, 2015). The results of this research prove that vitamin C can

fight against lung cancer and kill cancer cells to reduce tumor masses.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 5

Vitamin D3

Vitamin D, also known as the sunshine vitamin, is considered to be a prohormone that

controls calcium and phosphorus levels, maintains healthy bones and teeth, and provides a

defense against cancer, multiple sclerosis, type one diabetes, and other medical issues. This

nutrient comes in two forms: vitamin D2 and vitamin D3. In this senior thesis experiment,

Vitamin D3 will be used because this form is recommended and easy to obtain; the chosen form

of the vitamin comes from the sunlight’s conversion of cholesterol on the skin. Vitamin D3, also

known as cholecalciferol, is made before the sunlight converts cholesterol on the skin into

calcidiol, the active form of vitamin D (Ware, 2016). Additionally, vitamin D is proven to have

anticancer effects through its antiproliferative effects on the cell cycle. The sunshine vitamin

inhibits the cell cycle by increasing the expression of cyclin-dependent kinases (CDK) inhibitors

which stop cyclin-dependent kinases from allowing cell division and growth. Vitamin D

produces CDK inhibitors suppressing the growth and division of cancer cells (Edwards, 2015).

A group of scientists researched the effects of vitamin D3 and exercise routines on mice

with cancerous lung tumors. Their methodology consisted of gathering sixty mice diagnosed

with lung cancer and splitting them into four groups. Two groups received a low dose of vitamin

D3, and the other two control groups did not receive doses of the sunshine vitamin. Between the

groups consuming vitamin D3, one group was active by exercising on a running wheel while the

other group remained sedentary. Similarly, between the two control groups, one group was active

by exercising on a running wheel as well, and the other group did not exercise as much. Lung

size measurements were taken fourteen weeks later. The overall lung tumor area for the mice that

received vitamin D3 was about 14.1% smaller than the original size. On the other hand the lung

tumor area for the control groups was only 8.6% smaller (Williams, Hershberger, Personius, &
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 6

Ray, 2016). This study relates to our thesis because it proves that vitamin D3 has the ability to

kill cancer cells or decrease their cell area.

Hypothesis

Based on this research, vitamins C and D3 are each known to affect cancer cells. Vitamin

D3 is able to disrupt the cell cycle by stopping cell division and growth through the production

of CDK inhibitors. Vitamin C has also been shown to damage cancer cells through its hydrogen

peroxide damage. Therefore, it is reasonably expected that when vitamin C and D3 are

combined, the A549 cancer cells will decrease in size, and there will be a higher percentage of

dead cells.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 7

METHODOLOGY

Apparatus and Materials

BulkSupplements’s Vitamin C and Fisher Scientific’s D3 were used as our treatments

because research shows that these vitamin types could help fight cancer. The cancer used was the

A549 lung cancer cell line. Roswell Park Memorial Institute (RPMI) medium was used to put

vitamin C into powder form, and a pH probe was used to adjust its pH level. To make vitamin

D3 into a solution, ethanol was used as the solvent. Hydrogen peroxide was used as a positive

control because it is known that it kills cancer cells. Trypan blue was used to make the dead

cancer cells distinguishable by turning them blue. The treatments were put into six-well plates. A

hemocytometer was used to perform cell counts. Trypsin allowed the cancer cells to stop

adhering to the wells. An inverted microscope with a camera attached took random pictures of

the cells for the area calculations. The ImageJ computer program enabled the cells to be

measured by uploading pictures of the cells and then calculating their areas. Motorized pipettes,

rolling pipettes, and micropipettes were used with their corresponding tips to measure out

solutions. Inverted microscopes were also used to look at cell development and count.

Experiment Preparation

To prepare for the experiment, master stock solutions of Vitamin C, D3, and hydrogen

peroxide with concentrations of 100 micromolars were made for the A549 cells’ treatments.

Creating the vitamin C master stock solution, 0.881 grams of BulkSupplements’s pure ascorbic

acid (also known as vitamin C) powder was obtained and weighed in a weighing boat on a

balance, and 50 mL of Roswell Park Memorial Institute (RPMI) medium was added to dissolve

the powder in order to make a liquid solution. By adding sodium hydroxide (NaOH) and
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 8

measuring the pH level with a pH probe, the vitamin C solution was then neutralized from an

acidic pH to a pH of 7. Then, the treatment was filter sterilized with a complete sterile filter to

prevent contamination. Next, the vitamin D3 master solution was produced by adding 0.25 grams

of Fisher Scientific’s pure vitamin D3 powder into 6.5 mL of ethanol. Finally, the hydrogen

peroxide 100 micromolar solution was obtained by mixing 1 micro liter of hydrogen peroxide

with 10 mL of RPMI.

Procedure

Beginning the actual experiment, four six-well plates were obtained to hold the cells and

treatments with a final volume of 2 mL. The whole procedure was performed under a sterile

fume hood to prevent contaminations. 1 mL of the A549 cells (2x10^5 cells/mL) were each

distributed in 21 wells of the four six-well plates using a motorized pipette. Plate 1 consisted of

three no treatment wells for the negative control and three hydrogen peroxide wells for the

positive control. Using a rolling pipette, 1 mL of RPMI was added into the no treatment wells,

and 1 mL of the hydrogen peroxide master stock solution was distributed into the hydrogen

peroxide wells.

Plate 2 contained three wells for the vitamin C treatment at a concentration of 1

micromolar and three more wells for the other vitamin C treatment at a concentration of 2

micromolars. With a rolling pipette, 80 microliters of the 100 micromolar vitamin C master stock

solution was added into 4 mL of RPMI. Then, 1 mL of this new solution was micro pipetted into

three wells to have a final concentration of 1 micromolar of vitamin C. Next, 320 microliters of

the vitamin C master stock solution was added into 8 ml of RPMI. This new mixture was then

micro pipetted into the other three wells to have a final concentration of 2 micromolars of

vitamin C.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 9

Plate 3 consisted of three wells for the vitamin D3 treatment at a concentration of 1

micromolar and three more wells for the other vitamin D3 treatment at a concentration of 2

micromolars. Using a micropipette, 80 microliters of the vitamin D3 master stock solution was

added into 4 mL of RPMI to create a new stock solution. With a rolling pipette, 1 mL of the new

solution was distributed into three wells containing 1 ml of A549 cells to have a final volume of

2 mL and a final concentration of vitamin D3 at 1 micromolar. Next, 320 microliters was micro-

pipetted into 8 mL of RPMI to create a new stock solution again. 1 ml of the new stock was then

added into the other three wells of A549 cells to have a final volume of 2 ml and a final vitamin

D3 concentration of 2 micromolars.

The final plate, Plate 4, only had three wells of the vitamin C plus vitamin D3 treatment;

each had a concentration of 1 micromolar. Using the the vitamin C solution made for the 1

micromolar treatment for Plate 2, 0.5 mL was put into three wells containing the A549 cells with

a rolling pipette. Next, 0.5 mL of the vitamin D3 solution made for the 1 micromolar treatment

for Plate 3 was distributed into the same three wells with a rolling pipette. The final volume of

the three well was 2 mL in each well, and each vitamin had a final concentration of 1

micromolar.

Finally, all four plates were covered with the top of the six-well plates and labeled

depending on what treatment each well was in charge of. Next, they were put into a carbon

dioxide incubator for 48 hours. This environment allows the cells to not be affected by outside

sources and allows them to grow or to react to the treatments. After 48 hours, the cells were then

harvested and prepared for measuring data.

THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 10

Plate 1: Plate 2:

Plate 3: Plate 4:

Figure #1. Diagram of Treatment Placement

Measures

In order to obtain the results, each well had to be suspended. First, 100 microliters of

trypan blue was pipetted into microtubes, one for each well. The trypan blue dyes dead cells blue

and alive cells grey for a more accurate cell count. The microtubes were labeled to match each

treatment they contained. Next, 100 microliters from each well (excluding the no treatment) were

pipetted into the corresponding microtube by a 200 microliter micropipette. Using the same tip,

the solution was suspended up and down. The tips were changed between each well to prevent

contamination. Then, 12 microliters of the solution from the microtubes were pipetted onto a

chamber of a hemocytometer. The cells were counted from each microtube by counting the top

right, top left, center, bottom right, and bottom left quadrants of one chamber of the
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 11

hemocytometer. The total number of dead (blue) and alive (grey) cells were recorded. In order to

calculate the true cell count, each number of dead and alive cells, respectively, from each

treatment was multiplied by 2x2x10^3.

(Rogue, 2002)

For the No Treatment control group, trypsinization was necessary because the A549 cells

were still adhesive to the wells. They needed to be detached in order to have an easier time

counting cells and calculating their areas. The medium from the No Treatment wells were

pipetted into three separate tubes. After this, 0.4 microliter of trypsin were added to each No

Treatment well, which was left alone for five minutes. Once time was up, the wells were scraped,

and 1 microliter of medium was put back into the wells from the corresponding tubes. The

medium was then pipetted out of the wells and back into the tube, from which 12 microliter were

placed on a hemocytometer and were counted in the same manner as the other treatments.

After the cell counts, wet mounts were made by placing 8 microliters from each well onto

a glass slide. Using an inverted microscope with a camera attached, four pictures of cells were

taken randomly from each slide. The cell areas were then calculated using a program called

ImageJ. In ImageJ, the images were uploaded after having to save them on a USB. A measuring

scale was then created by opening a picture of 20x scale in millimeters. Next, a line was drawn
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 12

from the 0.1 mm to the 0.2 mm to set an appropriate scale for the area measurements. Then, the

freehand tool was selected to draw on the boundaries of the cells in order to measure their area.

ImageJ measures the area after the boundaries of the cell are drawn.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 13

RESULTS

Cell Area

A549 Cell Area Averages (mm)

3.50E-04

3.00E-04

2.50E-04

2.00E-04

1.50E-04

1.00E-04

5.00E-05

0.00E+00
No Treatment Hydrogen Vitamin C (1 Vitamin C (2 Vitamin D3 (1 Vitamin D3 (2 Vitamin C & D3
Peroxide um) um) um) um) (1 um each)

Vitamin Vitamin Vitamin C &

No Hydrogen Vitamin C (2 D3 Vitamin D3 (1 um
Treatment Peroxide C (1 um) um) (1 um) D3 (2 um) each)
3.22E-04 1.19E-04 1.53E-04 1.33E-04 1.59E-04 1.25E-04 1.42E-04
Figure #2. Cell Area Results

The charts above show the averages taken from the area of the cells affected by each treatment.
The cell areas are measured in millimeters.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 14

Cell Count

H2O2 No Vitamin Vitamin Vitamin Vitamin Vitamin

Treatment C C D3 D3 C and
1uM 2uM 1 uM 2 uM D3
1 uM
Trial Alive 0 48 17 6 10 4 1
1
Dead 56 0 16 53 10 83 34

Trial Alive 0 41 47 2 3 1 1
2
Dead 28 0 6 52 16 57 33

Trial Alive 0 45 48 4 1 4 3
3
Dead 37 0 17 39 28 61 17

Figure #3. Number of Dead and Alive Cells in Each Treatment.

The table above shows both the alive and the dead cell count in each trial. “uM” represents
“micromolar”. All numbers were then multiplied by 2x2x10^3 in order to create proper
estimations.

Percentage of Dead Cells

100
100 92.31 94.12 95.71 94.38
90
80
70
60
50
40
25.82
30
20
10 0
0
No Treatment H2O2 Vitamin C 1 Vitamin C 2 Vitamin D3 1 Vitamin D3 2 Vitamin C and
uM uM uM uM D3 1 uM

Figure #4. Percentage of Dead Cells in Each Treatment.

The graph above shows the percentage of dead cells out of the total cell count from the average
of all three trials for each treatment.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 15

DISCUSSION
How do varying combinations of Vitamin C and D3 affect cell viability and cell area of

A549 cells in cell cultures after being incubated for 48 hours? Previous research suggested that

the vitamins reduce tumor masses and inhibit the cell cycle of cancer cells. Because of these

studies, we hypothesized that vitamins C and D3, when combined, would decrease the size of the

A549 cancer cells and have a higher percentage of dead to alive cells.

Analysis of Data - Cell Area

The treatments and the positive control decreased the cancer cells’ areas while the no

treatment cells had the highest area. The positive control, the treatment of hydrogen peroxide,

decreased the area the most because hydrogen peroxide is very toxic and deadly to all cells;

therefore, it proved that the A549 cancer cells were capable of being affected from treatments.

However, our hypothesis was incorrect. The combination of vitamin C and D3 at 1 micromolar

each did not decrease the cell area the most, and it had results that were in between the results of

treatments with concentrations of 1 and 2 micromolars.

Nonetheless, our results proved that vitamin C and D3 both have the capabilities of

decreasing cancer cell sizes. Vitamin D3 at 2 micromolars decreased the cell area the most,

followed by Vitamin C at 2 micromolars and then the combination of vitamin C and D3 at 1

micromolar each. Vitamin D3 at 1 micromolar decreased the cell area the least, followed by

vitamin C at 1 micromolar. The treatments of 2 micromolars seem to decrease the area more

while the treatments with a concentration total of 1 micromolar did not do as well. Vitamin C

and D3 had very similar effects on the cells. Vitamin D3 decreased the cell area slightly more

when compared to treatments of the same concentration.

THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 16

Analysis of Data – Cell Count

The cell counts were partially as expected. To begin with, the no treatment wells had zero

dead cells, while the H2O2 treatment resulted in 100% dead cells. This is what was expected

because hydrogen peroxide is known to kill cancer cells. Therefore, the results from the positive

and negative controls show that the experiment was accurate. Overall, the vitamins were more

effective in the 2 micromolars solutions of each vitamin than in the 1 micromolar solutions. The

difference due to the micromolar level was especially significant for vitamin C. Between the two

vitamins, vitamin D3 was more effective than vitamin C. However, the vitamin C and D3

combination did not perform the best, as we had predicted. The only vitamin treatment that

functioned better than the combination was vitamin D3 at 2 micromolars. Thus, the reason the

combination did not work as well as expected may be because there was 1 micromolar of vitamin

D3, not 2 micromolars in the combination. Furthermore, the 1 micromolar of vitamin C worked

poorly when compared to the 2 micromolars of vitamin C, so only having 1 micromolar of

vitamin C in the combination may have caused further reason for fewer dead cells.

Vitamin C is already known to damage cells by breaking down into hydrogen peroxide,

while vitamin D3 is known to inhibit the cell cycle. Vitamin D3 may have left a higher

percentage of dead cells than vitamin C because vitamin D3 directly affects the life cycle of the

cell. Alternatively, there may have been catalase in the cancer cells that even at a low level could

have hindered the hydrogen peroxide produced by vitamin C from working as efficiently.

Limitations

Having this be a high school thesis, there are many limitations that could have affected

the results of this research. Time and money were a major constraint. Repeated trials of this
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 17

experiment would have increased the accuracy of our results; however, the equipment at Middle

Tennessee State University was for limited use and the materials used for the experiment were

expensive. More time would be needed to be able to collect all the materials again, but the thesis

experiment took about a month to learn how to use the equipment, to prepare the treatments, to

conduct the experiment, and to collect the data. Additionally, many of our measurements could

have not been very accurate due to the fact that most high schoolers are not accustomed to the

equipment in college laboratories.

THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 18

CONCLUSION

Overall, the results turned out to prove that both vitamin C and D3 have apoptotic effects

that inhibit A549 cells from growing. All the vitamin treatments decreased cell areas to an extent

and killed a high percentage of cells in a population. Unexpectedly, the combination did not

decrease the cell area the most and did not kill the most A549 cells. On the other hand, vitamin

D3 at 2 micromolars was the most effective to the cell area and the cell count.

In relation to cell area, the combination of vitamin C and D3 at 1 micromolar each was

only the third best out of the vitamin treatments. Some of the regular effects of vitamin C and D3

individually may have been inhibited due to forcing two vitamins to interact. Moreover, vitamin

D3 seemed to do better overall. Perhaps, the effect of vitamin D3 producing cyclin-dependent

kinases to suppress cancer cell growth was marginally more effective than the effect of vitamin

C producing hydrogen peroxide to kill cells. The vitamins treatments at 2 micromolars achieved

smaller cell areas than the treatments at 1 micromolar. Therefore, higher concentrations of

vitamin C and D3 are more effective when decreasing the area of A549 cells.

In relation to cell count, the vitamin C and D3 combination was the second most

effective, following vitamin D3 at 2 micromolars. The 2 micromolar treatments worked better

than the 1 micromolar treatments for both vitamins; therefore, the reason the combination did not

work as well may be due to using only one micromolar of vitamins C and D3 respectively, rather

than two. Alternatively, the effects may be due to vitamin D3 being hindered by vitamin C (as

vitamin C was not as successful), resulting in interference. Overall, each vitamin treatment did

kill cells, but vitamin D3 killed a larger percentage of cells than vitamin C or the combination

killed.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 19

For future use, the information of this thesis could be used for many cancer research

industries where vitamin therapy is being intensely studied. Vitamin therapy studies are

increasing in many cancer laboratories in the world today. If this experiment were to be further

investigated, testing normal, healthy cells to the treatments used in this thesis would extremely

help cancer research. If multiple trials were to be conducted to normal cells and A549 cancer

cells and the vitamin treatments proved to only be suppressing to the lung cancer cells, then there

would be another step made in finding the cure for lung cancer. Furthermore, finding this

information is very crucial because lung cancer affects many people today. Expanding cancer

research will only get the world closer to finding the cure that is yet to be found.
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 20

References
A549 (ATCC® CCL-185™). (n.d.). Retrieved November 14, 2017, from
https://www.atcc.org/Products/All/CCL-185
A549 Cell Line human 86012804. (n.d.). Retrieved November 14, 2017, from
http://www.sigmaaldrich.com/catalog/product/sigma/86012804?lang=en&region=US
Edwards, B. J. (2015, October 05). Anticancer Effects of Vitamin D. Retrieved December 10,
2017, from http://www.gotoper.com/publications/ajho/2015/2015oct/anticancer-effects-
of-vitamin-d

Fong, R., Ling, G., & Kah, M. (2015). Effects of High Doses of Vitamin C on Cancer Patients in
Singapore: Nine Cases [Scholarly project].

Gazdar, A. F., Girard, L., Lockwood, W. W., Lam, W. L., & Minna, J. D. (2010, September 08).
Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research. Retrieved
November 14, 2017, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935474/#bib25
Lerche, O. (2017, March 23). Vitamin C Could Treat Cancer: Dose of Substance Found in
Lemons Can Kill Deadly Cells. Retrieved December 10, 2017, from
https://www.express.co.uk/life-style/health/752822/cancer-disease-treatment-vitamin-C-
diet

Nordqvist, J. (2017, April 10). Vitamin C: Why is it important? Retrieved December 10, 2017,
from https://www.medicalnewstoday.com/articles/219352.php
PDQ Adult Treatment Editorial Board. (2017, November 9). Non-Small Cell Lung Cancer
Treatment. Retrieved November 14, 2017, from
https://www.cancer.gov/types/lung/patient/non-small-cell-lung-treatment-pdq
PDQ Adult Treatment Editorial Board. (2017, November 9). Small Cell Lung Cancer Treatment.
Retrieved November 14, 2017, from https://www.cancer.gov/types/lung/patient/small-
cell-lung-treatment-pdq
Removal of Adherent Cells from Culture Surface Using Trypsin. (n.d.). Retrieved November 14,
2017, from http://www.sigmaaldrich.com/technical-
documents/protocols/biology/removal-of-adherent-cells.html
Rogue, M. (2002, September 2). Retrieved March 5, 2018, from
http://www.vivo.colostate.edu/hbooks/pathphys/reprod/semeneval/hemacytometer.html
The American Cancer Society. (n.d.). Lung Cancer. Retrieved November 14, 2017, from
https://www.cancer.org/cancer/lung-cancer.html
The American Cancer Society. (n.d.). Lung Cancer Prevention and Early Detection. Retrieved
November 14, 2017, from https://www.cancer.org/cancer/lung-cancer/prevention-and-
early-detection.html
THE EFFECTS OF VITAMIN C AND D3 ON A549 CELLS 21

The American Cancer Society. (2016, February 8). Key Statistics for Lung Cancer. Retrieved
November 14, 2017, from https://www.cancer.org/cancer/non-small-cell-lung-
cancer/about/key-statistics.html
University of Iowa Health Care. (2017, January 9). Why High-dose Vitamin C Kills Cancer
Cells. Retrieved November 14, 2017, from
https://www.sciencedaily.com/releases/2017/01/170109134014.htm
Ware, M. (2017, November 13). Vitamin D: Health benefits, facts, and research. Retrieved
December 10, 2017, from https://www.medicalnewstoday.com/articles/161618.php

Williams, B. T., Hershberger, P. A., Personius, K. E., & Ray, A. D. (2016, April 01). The Effect
of Exercise and Vitamin D3 Supplementation on Tumorigenesis and Pulmonary
Function in a Lung Tumor Animal Model. Retrieved from
http://www.fasebj.org/content/30/1_Supplement/1167.6?related-
urls=yes&legid=fasebj%3B30%2F1_Supplement%2F1167.6