real extended essay

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

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, 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)

2ALLEYNE, D


TABLE OF CONTENTS

PAGE

Abstract …………………………………………………………………………………… 2

Rationale …………………………………………………………………………………… 4

Introduction

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

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

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

Hypothesis

Significance of Research

Materials and Method

Results

Data Processing

Discussion

Acknowledgements

Literature Cited

Appendix

3ALLEYNE, D


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 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.

4ALLEYNE, D


INTRODUCTION

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

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

1 (Merck 1996)2 (Reichlin S et al. Neuroendocrinology 217-218)

5ALLEYNE, D


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 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.

6ALLEYNE, D


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

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.

7ALLEYNE, D


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 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.

8ALLEYNE, D


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 to

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

attachment.

9ALLEYNE, D


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 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

10ALLEYNE, D


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.

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.

11ALLEYNE, D


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 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.

12ALLEYNE, D


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

LNCaP 24 Hours 48 HoursWell # 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 12 10 3 0 2 10 5 2 13 7 3 0 0 12 1 0 1

Average 9 4 0 1 12 3 1 1C4-2

Well # 0 mM 0.1 mM 1 mM 10 mM 0 mM 0.1 mM 1 mM 10 mM1 8 8 0 1 14 4 0 02 10 0 2 0 11 2 0 13 12 6 3 0 13 1 1 3

Average 10 5 2 0 13 2 0 1C4-2B 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 32 7 2 1 3 12 1 1 13 9 4 1 0 12 1 1 1

Average 8 3 1 1 14 1 1 2

13ALLEYNE, D


DATA PROCESSING

Table 2: Table of Raw Counts with Standard Deviations

LNCaP 24 Hours 48 HoursWell # 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 12 10 3 0 2 10 5 2 13 7 3 0 0 12 1 0 1

Average 9 4 0 1 12 3 1 1

SD1.73205

11.15470

1 0 11.52752

52.08166

61.15470

1 0C4-2

Well # 0 mM 0.1 mM 1 mM 10 mM 0 mM 0.1 mM 1 mM 10 mM1 8 8 0 1 14 4 0 02 10 0 2 0 11 2 0 13 12 6 3 0 13 1 1 3

Average 10 5 2 0 13 2 0 1

SD 24.16333

21.52752

5 0.577351.52752

51.52752

5 0.577351.52752

5C4-2BWell # 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 32 7 2 1 3 12 1 1 13 9 4 1 0 12 1 1 1

Average 8 3 1 1 14 1 1 2

SD1.15470

11.15470

1 0.577351.73205

14.04145

2 0.57735 0.577351.15470

1

14ALLEYNE, D


0 mM 0.1 mM 1 mM 10 mM0

2

4

6

8

10

12

14

24 h48 h

Concentration of Melatonin

Cell

Num

ber (

Cells

)

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

15ALLEYNE, D


0 mM 0.1 mM 1 mM 10 mM0

2

4

6

8

10

12

14

24 h48 h


Cell

Num

ber (

Cells

)

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

16ALLEYNE, D


0 mM 0.1 mM 1 mM 10 mM0

2

4

6

8

10

12

14

48 h24 h


Cell

Num

ber (

Cells

)

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

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.

17ALLEYNE, D

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