The Differential Effects of Melatonin on Growth

Inhibition in Human Prostate Cancer Cells

Research Question: Is there a differential effect on growth inhibition in the

human prostate cancer cell lines, C4-2 and C4-2B when they are treated with
melatonin?

Candidate Name: Dereck Antonio Shakeel Alleyne

Candidate Number: 000207-005

Subject: Biology Higher Level (Extended Essay)

Supervisor: Mr. Kris Wilson

Date: 20 December, 2010

 Candidate Number: 000207-005

Word Count:

ABSTRACT

Melatonin (N-acetyl-5-methoxytryptamine) is a serotonin derivative

hormone, secreted by the pineal gland in the brain. It is of particular interest
because although melatonin’s primary role lies in regulation of the body’s
circadian rhythm, much research has proven that it has anti-proliferative and
oncostatic effects on cancer cells. However, some types of cancer cells
respond better to melatonin than others. These types of cancers include
prostate cancer, the leading cause of death among men today. It is these
effects which lead to the question this experimental study seeks to
investigate. The question asks, Is there a differential effect on growth
inhibition in the human prostate cancer cell lines, C4-2 and C4-2B when
treated with melatonin?

LNCaP (androgen dependent - control), C4-2 and C4-2B (both androgen

independent), were cultured to 90% confluency in T-Medium. Stock flasks of
these cell lines were sub-cultured using 0.25% Trypsin-0.53 mM EDTA. Cells
were subsequently placed in experimental dishes, with T-Medium at a
density of 1 × 104 cells per ml, having only 1 ml of culture medium and cells
in each well. They were subsequently placed in an incubator. After 24 hours,
the T-Medium was removed and the cells were treated with one of four
concentrations of melatonin. The treatment groups included: 0 mM (control),
0.1 mM, 1 mM and 10 mM. The cells were then incubated another 24 hours,

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and then harvested and counted using a hemocytometer. This was also done
after 48 hours.

Using cell counts, the student t- test was performed and rendered that
melatonin has differential effects on these prostate cancer cell lines. Since p-
values were greater than 0.05, the null hypothesis was accepted, showing
that there was significance between the variables. It was also found that 1
mM melatonin was the most effective concentration for growth inhibition of
these cell lines.

(Word Count: 300)

TABLE OF CONTENTS

PAGE

Abstract ……………………………………………………………………………………

Rationale ……………………………………………………………………………………

Introduction

Background Information …………………………………………………………

Research Question ………………………………………………………………

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Experimental Variables …………………………………………………………….

Hypothesis

Significance of Research

Materials and Method

Results

Data Processing

Discussion

Acknowledgements

Literature Cited

Appendix

RATIONALE

The decision to complete an extended essay in biology comes from my

fascination with the life sciences. I always knew that I would pursue such a

research paper; however my research question was cultivated in my first

year spring semester IB Biology HL class. Ms. Escar Kusema spoke to my

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class about her research on x-organ neurite cell outgrowth of the Uca

Pugilator (Fiddler Crab), when these cells were treated with melatonin.

Immediately my interest sparked and I knew that I would pursue an essay

which investigated melatonin’s physiological actions. It is not every day that

one comes across such a hormone like melatonin. My primary research leads

me to the area of melatonin’s action on cancer. Knowing that I live in a

country where little to no research surrounding cancer is ever done, lead me

deeper. Prostate cancer is one of the cancers which are the cause of death

among many men in Barbados. However, seeing that melatonin had a

special action on melatonin via androgen receptors solidified my topic. The

use of C4-2 and C4-2B cell lines, are models of cancer commonly presented

in Barbados, since men over 50 years of age present in this stage. Therefore,

this research focuses on the molecular actions of melatonin on these cancer

cells which can then possibly help find a cure for this disease.

I contacted Dr. Carol Linder at New Mexico Highlands University, and she

graciously lent her facilities and time to ensure that my project was a

success.

INTRODUCTION

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Background Information

Melatonin, (N-acetyl-5-methoxytryptamine) is a hormone of the pineal gland,

also produced by extra-pineal tissues (Merck 1996)1. This hormone mainly

regulates the body’s circadian rhythms but research has proven that it plays

a major role in the regulation of many physiological and psychological

processes2. Melatonin has proven to be a strong anti-oxidant, and has been

shown to produce oncostatic and anti-proliferative effects.

What is interesting about melatonin is that it has been shown to be very

effective in some cancers(Ann W. Hsing, 2010). These cancers include

prostate cancer, which is the primary cancer type in this study. It has been

shown that in prostate cancer the serum levels of melatonin are lower than

normal. Prostate cancer is very interesting in terms of how it proliferates

within the body. The cancer starts out as androgen dependent. Prostate

cancer depends highly on testosterone to proliferate. What happens is that

testosterone binds to the androgen receptor located in the cytosol of the

prostate cancer cell and changes it to a derivative of testosterone known as,

dihydrotestosterone, which allows for the proliferation of the cells. However

as scientist found that testosterone was necessary, they created drugs which

1 (Merck 1996)

2 (Reichlin S et al. Neuroendocrinology 217-218)

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blocked testosterone from binding to the androgen receptor. This seemed

like a reasonable treatment for combatting the cancer, but to their

amazement the cancer did not die, but became more aggressive. What the

prostate cancer cells do is that when hormone ablation therapy is occurring,

they lose their dependence on androgen (testosterone) but they still express

the androgen receptor.

The research done on melatonin and its relationship to prostate cancer help

us to understand it’s mechanism for proliferation. It is postulated that

melatonin’s action in actively proliferating prostate cancer is directly

associated with the androgen signaling axis (ASA). Thus when melatonin

interacts with ASA it affects the cell cycle, leaving cells in the G1/G0 phase.

Looking at the cell cycle and what occurs, we then see that at those phases

DNA replication occurs. However melatonin stops the cells from maturing

any further and so can be said to induce cell death or apoptosis. It has been

found that melatonin at nano-molar levels have quite an effect on prostate

cancer.

The cell lines used in this study actively models the progression of human

prostate cancer, from its androgen dependence to its androgen

independence. LNCaP is the androgen dependent cell line which had

metastasized to the lymph node, which it derivative sublines C4-2 and C4-2B

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are both androgen independent and have metastasized to the bone. The

sublines C4-2 and C4-2B are very unique in that they have similar genetic

backgrounds but differ immensely in their androgen independence and

metastatic potential. Using melatonin treatments on these cell lines seeks to

understand if there is differences within the growth-inhibition effect and how

these differences can lead to finding treatments which can be used to treat

this cancer.

Research Question

This experimental study seeks to investigate the following research question:

“Is there a differential effect on growth inhibition in the human prostate

cancer cell lines, C4-2 and C4-2B, when they are treated with melatonin?”

This question then allows us to answer another question, which is, “At what

concentration is melatonin effective in inhibiting growth in these prostate

cancer cell lines?”

Experimental Variables

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Dependent: The concentrations of melatonin were changed.

Independent: The cell growth inhibition and viability was in response to the

changes in concentration of melatonin.

Controlled: The length of time for incubation, the medium used and the

conditions for incubation were controlled. The seeding density of the cells

was also kept constant. Also the control of substances (melatonin) that the

cells were exposed to were also kept constant, which included no extra

serum or additives for the growing cells. They were treated all the same.

Hypothesis

Melatonin will not have a differential growth inhibition effect on C4-2 and C4-

2B, since they are isogenic cell lines, and therefore expression of genes and

genetic backgrounds are similar or the same. Since the androgen receptor

gene is expressed in both cell lines, and the androgen receptor is the binding

site for melatonin, the growth inhibition effect would not be significant.

Significance of Research

Cancer will always be known as the debilitating disease that takes many

lives in the world. For me, my research is significant really to the Caribbean;

especially to my little island Barbados. The reason I chose to work with the

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C4-2 and C4-2B human prostate cancer cell lines is because these are

representative of the stages of prostate cancer which present throughout the

Caribbean. Many men who are afraid to have prostatic examinations done by

their physicians or those who can’t afford to have it done are the ones who

present with prostate cancer which has metastasized into the bones. My

research is significant since it aims to study the molecular events that occur

as melatonin interacts with prostate cancer. Although much research is

being done on melatonin’s interactions with various cancers, much is not

understood about the C4-2 and C4-2B stages of prostate cancer. My research

then serves as a primer to begin to study these stages of prostate cancer

which affect many men in the Caribbean. My research only seeks to

investigate if melatonin will in fact halt cellular proliferation, and then from

there seeking to understand how it does this.

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Materials and Method

Cell Culture

LNCaP, C4-2 and C4-2B, human prostate cancer cell lines were cultured and

maintained in T-Medium (growth medium), supplemented with 100 u/ml of

penicillin-streptomycin solution in T-75 cell culture flasks. The cultures were

incubated at 37oC in 5 % CO2. Stock flasks were then randomly selected, and

growth medium was removed followed by washing the cell monolayer with 5

ml Phosphate Buffered Saline (PBS) solution, then careful passaging using

0.5% Trypsin-0.53mM EDTA in1 × Hanks Balanced Salt Solution (HBSS). The

cells were incubated for approximately five (5) minutes following this

procedure. After, cells were manually dislodged from the surface of the flask

by using a cell scraper. 5 ml growth medium was added to stop the

trypsinization process. The newly made cell suspension was then centrifuged

at 200 G for five minutes. The supernatant was removed and the cell pellet

that formed was then re-suspended in 12 ml growth medium. A 1 ml sample

of the cell suspension was removed for hemocytometer counts. When the

cell number was determined, cells were adjusted to 100,000 cells per ml, by

adding growth medium as a dilution. 1 ml of the cell suspension was added

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to each well of a 12 well cell culture plate, with 3 ml of culture medium to

allow for cellular attachment.

Preparation of Melatonin Solutions

Since melatonin is insoluble in water, the solvent used was 70% ethanol.

Calculations were done to find the mass necessary to make a 10 mM stock

solution of melatonin. 0.02323 g of melatonin powder (MW = 232.3 g/mol)

was diluted in 1 ml of 70% ethanol and stirred vigorously. Subsequently,

dilutions to achieve required treatment concentrations were done by diluting

the stock solution with the culture medium. This procedure was repeated to

achieve enough of the melatonin solution for each trial of the experiment.

Treatments

Twenty-four (24) hours after cells were plated; the growth medium was

removed and replaced with growth medium, containing one of the four

treatment groups. The treatment groups were: 0 mM melatonin as the

control, 0.1 mM, 1 mM, and 10 mM melatonin. The cell plates were placed in

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the incubator and allowed to grow for a period of 24 and 48 hours where

they were harvested for growth studies. By splitting the cells into two 12 well

plates per cell line, each concentration could be done as a triplicate, which

renders more significant data because of the sample size and in turn reduces

the percentage error for the entire study.

Growth Studies

Cells were harvested beginning on day 1 after the addition of melatonin. The

cell plate to be analyzed for growth was removed from the incubator. The

medium was removed, and 1 ml of phosphate buffered saline (PBS) solution

was used to wash the cell monolayer. This was important since serum

components in the growth medium can disrupt the harvesting process by

inhibiting trypsin activity. Subsequently, 1 ml of 0.5% Trypsin-0.53 mM EDTA

in 1 × HBSS was added to the well of interest, and allowed to incubate for 5

minutes. After, 1 ml of growth medium was added. The cell suspension was

harvested, and centrifuged at 200 G for 5 minutes. The supernatant was

removed and the cell pellet was re-suspended in 2 ml growth medium. A 1

ml sample of the cell suspension was removed and placed in an Eppendorff®

tube, for later counts with the hemocytometer. The remainder of cell

suspension was fixed in 25% paraformaldehyde and stained with 0.5%

crystal violet in methanol.

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Counting Cells

The hemocytometer was prepared by wiping the surface with 70% ethanol

solution. Using a P10 micropipette, 10µl of Diethyl Pyrocarbonate (DEPC)

treated water was transferred to the edges of the hemocytometer, in small

drops. A large glass cover slip was then fixed onto the surface of the

hemocytometer. The water was used to keep the cover slip in place while

counting the cells.

A stock solution of 0.1% Trypan Blue in PBS was made by transferring 250µl

of 0.4% Trypan Blue, to 750 µl of PBS. This was made in a 50 ml conical tube

and then stirred vigorously to ensure proper mixing. 20µl of the cell

suspension was placed into a microfuge tube, and mixed with 20µl of 0.1%

Trypan Blue in PBS. The hemocytometer was loaded using a P10

micropipette. 20µl of cell suspension in 0.1% Trypan Blue in PBS was loaded

in one chamber of the hemocytometer, by pressing the tip of the pipette,

into the “V” groove on the chamber. The cell suspension in Trypan Blue was

expelled and allowed to be drawn into the chamber by capillary action.

Using the 10 × objective lens of the inverted microscope, the grid for the first

chamber was observed. Viable cells were counted only, in the four corners of

the grid, using a manual tally counter. Viable cells were distinguished from

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dead cells, by their appearance under the microscope. Viable cells were

transparent, however dead cells were blue and stained with the Trypan Blue

solution. This was also done for the second chamber. An average of the cell

numbers was found and the following equation was used to determine the

number of cells:

Cells/ml = # cells counted/4 (# quadrants) × 4 (Dilution Factor) × 104

Total Cells = Cells/ml × Total Original Volume of Cell Suspension from

which sample was taken.

After, the hemocytometer was decontaminated using 70% ethanol.

Statistical Analysis

The data obtained was expressed as both raw data cell counts and the arithmetic

mean ± Standard Deviation. When cells were counted, the averages found from the

two chambers of the hemocytometer were expressed to the nearest whole number

for clarity of results. Also the student t-test, unpaired was used to analyze the

significance difference between the cell numbers of the control experiment versus

the cell numbers of the experiments with melatonin. Also the student t-test was

used to test the significance difference between each of the treatment groups. P

values < 0.005 were considered statistically significant and the null hypothesis was

rejected. P-values > 0.005 were considered statistically insignificant and the null

hypothesis was accepted.

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RESULTS

The following table shows results of the raw cell counts, and do not represent

the actual number of the cells counted. The number of the cells which were

counted were kept to their raw count for clarity of results.

Table 1: Raw Cell Number Counts for 24 hours and 48 hours

LNCa
P 24 Hours 48 Hours
Well # 0 mM 0.1 mM 1 mM 10 mM 0 mM 0.1 mM 1 mM 10 mM
1 10 5 0 1 13 2 0 1
2 10 3 0 2 10 5 2 1
3 7 3 0 0 12 1 0 1
Avera
ge 9 4 0 1 12 3 1 1
C4-2
Well # 0 mM 0.1 mM 1 mM 10 mM 0 mM 0.1 mM 1 mM 10 mM
1 8 8 0 1 14 4 0 0
2 10 0 2 0 11 2 0 1
3 12 6 3 0 13 1 1 3
Avera
ge 10 5 2 0 13 2 0 1
C4-2B
Well # 0 mM 0.1 mM 1 mM 10 mM 0 mM 0.1 mM 1 mM 10 mM
1 9 4 2 0 19 0 0 3
2 7 2 1 3 12 1 1 1
3 9 4 1 0 12 1 1 1
Avera
ge 8 3 1 1 14 1 1 2

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DATA PROCESSING

Table 2: Table of Raw Counts with Standard Deviations

LNCa
P 24 Hours 48 Hours
Well 0.1 0.1
# 0 mM mM 1 mM 10 mM 0 mM mM 1 mM 10 mM
1 10 5 0 1 13 2 0 1
2 10 3 0 2 10 5 2 1
3 7 3 0 0 12 1 0 1
Avera
ge 9 4 0 1 12 3 1 1
1.732 1.154 1.527 2.081 1.154
SD 051 701 0 1 525 666 701 0
C4-2
Well 0.1 0.1
# 0 mM mM 1 mM 10 mM 0 mM mM 1 mM 10 mM
1 8 8 0 1 14 4 0 0
2 10 0 2 0 11 2 0 1
3 12 6 3 0 13 1 1 3
Avera
ge 10 5 2 0 13 2 0 1
4.163 1.527 0.577 1.527 1.527 0.577 1.527
SD 2 332 525 35 525 525 35 525
C4-
2B

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Well 0.1 0.1

# 0 mM mM 1 mM 10 mM 0 mM mM 1 mM 10 mM
1 9 4 2 0 19 0 0 3
2 7 2 1 3 12 1 1 1
3 9 4 1 0 12 1 1 1
Avera
ge 8 3 1 1 14 1 1 2
1.154 1.154 0.577 1.732 4.041 0.577 0.577 1.154
SD 701 701 35 051 452 35 35 701

Graph 1: Concentration of Melatonin vs. Cell Number– LNCaP

Graph 2: Concentration of Melatonin vs. Cell Number– C4-2

Graph 3: Concentration of Melatonin vs. Cell Number– C4-2B

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The error value for each of these graphs is equal to the standard deviation of each

cell line, for all concentrations of melatonin and for each time period.

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