Stauber

Ef f e c t s of Ch e m ica l s i n Cruci f e rous

Ve ge ta b le s on M a m m a ry Ca rci nom a

Sarah Joy Stauber

January 9 March 2, 2014
Niles North High School
Mrs. Camel
Period 9
1
(National Cancer Institute, 1989)

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Ta b l e o f Con t e n t s
Title Page...........................................................................................................................................................................1
Table of Contents......................................................................................................................................................2
Acknowledgements..................................................................................................................................................3
Purpose, Hypothesis & Rational..................................................................................................................4
Review of Literature................................................................................................................................................5
Materials..........................................................................................................................................................................10
Procedure........................................................................................................................................................................13
Variables..........................................................................................................................................................................22
Results..............................................................................................................................................................................24
Statistical Analysis..................................................................................................................................................27
Experimental Error................................................................................................................................................29
Conclusion......................................................................................................................................................................31
Reference List............................................................................................................................................................33

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I would like to acknowledge my teachers and friends who have both helped me with my
paper and project in the past weeks. First and foremost, I thank Mrs. Camel for being
patient with me and helping me with everything from editing the beginnings of my paper
to putting the finishing touches on my data collection. I would also like to thank Ms.
France for patiently walking me through properly analyzing data and helping me further
understand my project. Additionally, I am very grateful to Mrs. Camel, Mr. Thielson and
Mrs. Posnock for staying long after school and coming in on the weekends to help make
sure my project was a success. I'd also like to give thanks to Maggi who showed me how to
properly work with cancer cells, and Mia for being so willing to share her cancer cells with
me and patient through my endless questions. Especial thanks to my good friends,
Elizabeth, Ambria and Mia, whom have given me moral support and helped me keep on
track in the past months. Lastly, I would thank my parents who have helped me get rides
to and from school on my days off and helping with the tiny details to make my project
just perfect.
Thank you!

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Purpose:
Can the chemicals in cruciferous vegetables directly cause apoptosis in breast cancer
cells? If so, which form is most effective: original glucosinolate or decomposed (by
myrosinase) isothiocyanate?

Hypothesis:
If sinigrin and allyl isothiocyanate cause apoptosis in breast cancer tissue, and allyl
isothiocyanate induces more, then it would be advisable to consume more raw cruciferous
vegetables to prevent the risk or treat of breast cancer. If neither reacts, then it can be
deduced that raw glucosinolate and/or isothiocyanate must be digested and processed
through the immune system before being able to have cancer-preventative properties.

Rational:
Researchers have been baffled by the cause of such cancer-preventative strength
from isothiocyanates and glucosinolates for decades. If we can determine that it is or is
not caused by direct contact, that could narrow down the possibilities. As for
differentiating between glucosinolate sinigrin and allyl isothiocyanate, the former is
naturally found in raw vegetables, but is catalyzed by myrosinase, so when the bonds are
broken, it turns into the later allyl isothiocyanate. In order to keep the original sinigrin
hydrate, the catalyst must be decomposed by heat, but not to the point where the
glucosinolate is also destroyed.

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Review of Literature:
In recent times, breast cancer has become an increasingly prominent issue,
accounting for 22.9% of all cancers worldwide and in 2008 alone, killing 458,503 women.
That is, attributing 13.7% of all cancer deaths worldwide to breast cancer (Globocan, 2010).
Areas in the world with the highest diagnoses rates
include North America, Nordic Europe, England, France,
and Australia, as shown in figure 1 (Worldwide Breast
Cancer, 2009). One recent statistic showed that out of
every 100,000 Caucasian women in the United States, 113.2 Figure 1: Number of new breast cancer
developed breast cancer; making breast cancer the most

cases occurring worldwide, per 100,000

people (Worldwide Breast Cancer, 2009)

prominent cancer among American women. While many studifigues show that cases of
diagnosed cancer are increasing, the survival rates have likewise increased. One study
shows that survival has increased six percent from 74% between 1974 and 1976 to 80%
between 1983 and 1990 (Cutter, Sigstedt, & Venne, 1999).
Cancer does not simply happen: it forms, it grows, and if caught too late, it
metastasizes. Of the roughly 37.2 trillion cells in the human body (Zimmer, 2013), there is
occasionally a mutation that causes a cell to change shape or behavior, and thus change
function. Normally, cells have a function called apoptosis in which upon realizing a
replication error, commit cell suicide. But when this doesn't happen, the mutant cell will
often continue dividing and replicating. When this excessively replicated mass grows into
surrounding tissue, it becomes a neoplasm, or tumor. Sometimes the growth will slow and
simply remain a benign tumor, or carcinoma in sitio, for the duration of its existence.

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However, if its growth does not slow and spreads into the bloodstream, the neoplasm
moves into the stage of metastasis. At which point, the newly malignant tumor that was
once isolated to one part of the body will start appearing in other places (Cutter, Siggstedt,
& Venne, 1999). Specifically breast cancer is known to spread into the bone, brain, liver and
lung (National Cancer Institute, 2013). If it is allowed to spread unchecked, the tumor will
overwhelm and exhaust the body's vital organs, eventually killing them and their host
(Cutter, Siggstedt, & Venne, 1999).
While benign tumors may remain for as long as a lifetime without further growing,
they should still be checked by a doctor due to their increased risk of nevertheless
becoming malignant (National Cancer Institute, 2005). On
the other hand, a malignant tumor can also form out of an
Figure 2: Different types of tumors as
evolve into malignancy (Cutter,
Siggstedt, & Venne, 1999)

abnormally dense pack of cells that have simply

replicated a little faster than usual, also known as

hyperplasia. While many go away on their own, some continue to grow into dysplasia, the
only difference being that they no longer line up in straight rows. If the mass continues to
grow into neighboring tissue, it gains the new classification of carcinoma in sitio, in which
case it is also like any other neoplasm & significantly gains the likelihood of becoming
malignant, as shown in figure 2 (Cutter, Siggstedt, & Venne, 1999).
Most tumors, however, are caused by certain chemicals, radiation (UV, gamma, etc.),
DNA-altering viruses and bacteria, genetics, diet, and hormones. All of these known
carcinogens work by altering the genetic code of human cells through one method or
another. Some of these factors people have power to change, but many are out of the

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common man's hands. For example, while it is globally possible to move from places that
have chemical or radiation contamination, it is often difficult to know if there is any in the
first place, and if so, how long it has been there. Even though one can say if a relative has
died due to cancer, there is little to do about changing genetics. All that can be done is
ensuring regular check-ups. On the other hand, people do have control over whether or not
they smoke. Chemicals like aminostilbene, arsenic, cadmium, chrysene, polonium-210, Ndibutylnitrosamine and S-methylfluoranthene found in cigarettes have shown strong
correlations to one's risk of developing cancer. One study has shown that there is about a
20-year lag from smoking a first cigarette to developing cancer. Besides that, the more one
smokes, the sooner and more likely that cancer will develop. Another cause of cancer is
related to UV exposure. Studies have shown that in cities with more days of sunshine,
there are also more cases of cancer. There are some viruses associated with human
cancers: HIV can lead to Kaposi's sarcoma, hepatitis B is related to liver cancer, human
papillomavirus is known to cause cervical cancer, and human T-cell lymphotrophic virus can
cause adult T-cell leukemia (National Cancer Institute, 2005).
Some of the more well known treatments include chemotherapy, surgery, and
radiotherapy (IQWiG, 2009). But there are others that have shown to be just as (if not more)
effective than these traditional therapies. One of them being the simple consumption of
cruciferous vegetables (broccoli, kale, green cabbage, etc.) One study done at Johns
Hopkins School of Medicine, testing the effects of cruciferous vegetables on chances of
developing cancer showed that the test group that had dithiocarbamates in their urine 8-24
hours after eating some veggies, also had a much lower risk of developing cancer than the

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experiment's control group that was not permitted any such vegetables. Despite all the
research and experimentation, however, it is still unknown as to how or why vegetables
have such an effect (Shapiro, Fahey, Wade, Stephenson, & Talalay, 1998). Some believe that
the chemicals in cruciferous vegetables induce phase II enzymes that protect against
carcinogenesis (spreading of cancer) by increasing water solubility and elimination (Higdon
& Drake, 2008). But others say that, "Isothiocyanates, derived from glucosinolates, are
thought to be responsible for the chemoprotective actions conferred by higher cruciferous
vegetable intake" (Navarro & Lampe, 2011).
Fresh cruciferous vegetables consist mainly of glucosinolates (sulfur-containing
chemicals, which when bonds are broken and
Figure 3: The chemical process ofglucosinolates
decomposing intoisothiocyanates. (Btheta, 2012)

the enzyme myrosinase released, transforms

into an isothiocyanate, as shown in figure 3.

Leafy greens that are cooked at a high enough temperature also have much higher
quantities of glucosinolates upon consumption because the heat deactivates and kills the
myrosinase catalyst. Different forms of each of these chemicals have shown to have
differing effects in cancer research, but the difference is minimal (Fahey, Zhang, & Talalay,
1997). Specifically glucosinolate sinigrin hydrate and allyl isothiocyanate (AITC) are the most
common forms of these chemicals, found in horseradish, mustard, radish, broccoli, Brussels
sprouts, and cabbage (Higdon & Drake, 2008).
For an even more concentrated source of such chemicals, one research group tested
and compared the potency of 3-day-old broccoli sprouts against mature flowers. The
findings show that tthe sprouts have 10-100 times the concentration of glucosinolates than

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mature cruciferous vegetables. Tested on mice with cancer, the tumor incident rate is
significantly lower in rats given higher concentrations of both glucosinolates &
isothiocyanates. Thus chemicals from cruciferous vegetables have the ability to slow, if not
stop, cancer replication. Besides that, they also found that eating fresh broccoli is eight
times as potent as frozen (Fahey, Zhang, & Talalay, 1997).
Given all the highly invasive treatments for cancer, it makes one think if there is
some more natural, easier method by which to remove or even prevent developing cancer
in the first place. That's where cruciferous vegetables come into play, offering a
preventative treatment method for most cancers. Why endure chemotherapy and/or
surgery when the chances of preventing cancer can be increased by simply eating
vegetables? The differing arguments regarding how glucosinolates and isothiocyanates
function, however, bring doubt into one's mind as to their actual effectiveness. It may be
possible that they directly cause the cells to die, but other research supports that these
chemicals work through the body's immune system. Either way, it would be helpful to
identify how and why they function.

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Materials:

disposable gloves (small)

invasive ductal carcinoma (breast cancer)

500 mL DMEM (High Glucose with LGlutamine Sterile-filtered) plain media

37 C water bath

30 mL trypsin

4X 2.0 mL cryovial

25 mL 100% Fetal Calf Serum (FCS)

100 mg insulin

2.5 mL Penstrep

Thermo Scientific (Mr. Frosty)

BIOHIT midi plus electric pipetting pump

10 mL graduated pipette tips

31X biolete 75cm3 culture flask

CO2 incubator (36.5 C & 5.0% CO2)

inverted microscope

70% ethanol in a spray bottle

paper towels

5X test tubes

eppendrof Centrifuge 5702

complete media (250 mL DMEM + 100 mg insulin + 25 mL FCS + 2.5 mL Penstretp)

bio-hood

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3M whole face carbon filter gas mask

designated cell-disposal bag

designated cell waste container

test tube rack

lab coat

fume hood

7X 10 mL erlenmeyer flask

permanent marker

distilled water

0.5-10 L micropipette

500-1000 L micropipette

sterile aerosol pipet tips for 10 L pipettor

sterile aerosol pipet tips for 100 L pipettor

0.98 L C4H5NS (Allyl Isothiocyanate) from mustard

laboratory film parafilm

OHAUS electronic analytical balance

metal scoop

2X weigh boat

3mg C21H36O (Urushiol) from poison ivy

4mg C10H16KNO9S2 xH2O (sinigrin hydrate) from horseradish

1X 24 microplate

1 mL graduated pipette tips

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Clay Adams hemocytometer

2-40 L micropipette

20-200 L micropipette

sterile aerosol pipet tips for 200 L pipettor

sterile aerosol pipet tips for 40 L pipettor

Premiere optical lens paper (4 X 6)

HyClone Trypan Blue Solution (0.2 um Filttered)

Boreal max. 100X magnification electronic microscope

Hand Tally Counter

bleach spray

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Procedure:
Part A: Preparing cells for storage (freezing at -80 C)
1.

Begin by putting the complete and plain media into a water bath at 37 C.

2. Thaw the trypsin by putting it into the CO2 incubator.

3. Remove media from flask and pour into waste container.
4. Pour (if already aliquoted) or pipette 2 mL of trypsin into flask over cells.
5.

Incubate the flask at 37 C in the incubator for about 10 minutes.

6. Examine the cells under the inverted microscope to determine if the cells have
released from the plastic.
7. Gently smack the flask on the work surface of the hood to help release the cells.
8. Pipette the liquid, now containing the cells, into a labeled test tube.
9. Wash the flask by pipetting the 6 mL of PLAIN media into the flask and combine the
wash with the media and cells in the test tube.
10. Centrifuge the test tube at 1000 RPMs for 10 minutes.
11. Micropipette out the media into the waste container.
12. Leave pellet at bottom of the test tube and add 0.5 mL of 100% Fetal Calf Serum
(FCS). Mix by pipetting up and down.
13. Transfer the mixture into a 2.0 mL cryovial.
14. Add 0.5 mL of 20% DMEM (diluted with plain RPM). Drip it in a drop at a time while
hand mixing.
15. Mix with a pipette and put in the Mr. Frosty.
16. Freeze at -80 C.

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Part B: Breast Cancer Culturing

1.

Begin by putting the complete and plain media into a water bath at 37 C.

2. Thaw the trypsin by putting it into the CO2 incubator.

3. Remove media from flask with an electric pipetting pump and 10 mL graduated
pipette tip and pour into waste container. Dispose tip.
4. Pour (if already aliquoted) 2 mL of trypsin into flask over cells.
5.

Incubate the flask at 37 C in the incubator for about 7 min.

6. Examine the cells under the inverted microscope to determine if the cells have
released from the plastic.
7. If they havent released, gently smack the flask on the working surface of the hood
to help release the cells.
8. Rub gloves with 70% ethanol to kill possible contaminating bacteria.
9. With a new tip, pipette the liquid, now containing the cells, into a labeled test tube.
10. Wash the flask by pipetting 3 mL of PLAIN media into the flask and combine the
wash with the media and cells into the test tube.
11. Centrifuge the test tube at 1000 RPMs for 10 minutes. Remember, the centrifuge
needs to be balanced.
12. Remove the media with the electronic pipetting pump and a new tip into the waste
container.
13. Re-suspend the cells by adding approximately 7 mL of complete media to the test
tube with the pellet of cells and mix by pipetting up and down.
14. Pipette 3.5 mL of cells into each of two biolite 75 cm2 culture flasks.

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15. Pipette another 2 mL of complete media into each culture flask.

16. Put into the incubator.
17. Dispose empty culture flask and test tube in designated cell disposal bag.
18. Clean the bio-hood with 70% ethanol, both the inside of the glass and work surface.
UV the bio-hood at the end of each day.
19. Repeat steps 1-18 each time the cells meet 50-80% confluency (which can take 2-5
days) for each culture flask until 15 separate culture flasks have been used.

Part C: Making Chemical Solutions

1.

Put on whole face carbon filter gas mask and lab coat.

2. Turn on and place all chemicals under a fume hood.

3. Fill a 100 mL beaker with roughly 600 mL of distilled water.
4. Create a 1000 M stock solution of Allyl Isothiocyanate.
a. Label a 10 mL erlenmeyer flask with a permanent marker: Allyl Isothiocyanate.
b. Using a 0.5-10L micropipette, add 0.00098 (0.001) mL of C4H5NS to the
erlenmeyer flask. Dispose of micropipette tip.
c. Using a 500-1000L micropipette, add water from 100 mL beaker until the 10
mL line is reached.
d. Mix the solution by pushing up and down with the micropipette. Dispose
micropipette tip.
e. Parafilm the top of the flask.
f.
5.

Carefully close bottle cap and replace in protective wrappings.

Create a stock solution of Sinigrin

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a. Label a 10 mL erlenmeyer flask with a permanent marker: Sinigrin

b. Using an electronic analytical balance, metal scoop and another weigh boat,
add exactly 0.0040g of C10H16KNO9S2xH2O to the flask
c. Using a new tip, fill the erlenmeyer flask until it is full of 10 mL of distilled
water with the 500-1000L micropipette.
d. Mix the solution by pushing up and down with the micropipette. Dispose
micropipette tip.
e. Parafilm the top of the flask.
f.

Carefully close bottle cap and replace in protective wrappings.

6. Label another 10 mL erlenmeyer flask with a permanent marker: H2O. With 5001000L micropipette, pipette distilled water until filled to 10 mL line.
7. Create 100 M solution of allyl isothiocyanate (serial dilution)
a. Label a 10 mL glass beaker with a permanent marker: Allyl Isothiocyanate
100M.
b. Fill the erlenmeyer flask with 1 mL (1000L) of allyl isothiocyanate stock
solution using 500-1000L micropipette. Dispose micropipette tip.
c. Using a new tip, fill the erlenmeyer flask until it is full of 10 mL of distilled
water with the 500-1000L micropipette.
d. Parafilm the top of the flask.
8. Create 10 M solution of allyl isothiocyanate (serial dilution)
a. Label a 10 mL glass beaker with a permanent marker: Allyl Isothiocyanate
10M.

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b. Fill the erlenmeyer flask with 1 mL (1000L) of 100 M allyl isothiocyanate

solution (from step 8) using the 500-1000L micropipette. Dispose tip.
c. Using a new tip, fill the erlenmeyer flask until it is full of 10 mL of distilled
water with the 500-1000L micropipette.
d. Parafilm the top of the flask.
9. Repeat steps 8-9 for the control (water) and sinigrin.

Part D: Preparing Cells for Testing

1.

Begin by putting the complete and plain media into a water bath at 37 C.

2. Thaw the trypsin by putting it into the CO2 incubator.

3. Prepare 5 culture flasks for storage (follow steps in Part A)
4. Remove media from flask with an electric pipetting pump and 10 mL graduated
pipette tip and pour into waste container. Dispose tip.
5.

Pour (if already aliquoted) 2 mL of trypsin into flask over cells.

6. Incubate the flask at 37 C in the incubator for about 7 min.

7. Examine the cells under the inverted microscope to determine if the cells have
released from the plastic.
8. If they havent released, gently smack the flask on the working surface of the hood
to help release the cells.
9. Rub gloves with 70% ethanol to kill possible contaminating bacteria.
10. With a new tip, pipette the liquid, now containing the cells, into a labeled test tube.
Dispose pipette tip.
11. With a new tip, wash the flask by pipetting 3 mL of PLAIN media into the flask and

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combine the wash with the media and cells into the test tube. Dispose tip.
12. Centrifuge the test tube at 1000 RPMs for 10 minutes. Remember, the centrifuge
needs to be balanced.
13. Remove the media with the electronic pipetting pump and new tip into the waste
container.
14. Re-suspend the cells by adding approximately 4 mL of complete media to each of 3
test tubes with the pellet of cells and mix by pipetting up and down.
15. Set aside test tubes in a test tube rack for Part E.
16. Dispose empty culture flask and test tube in designated cell disposal bag.
17. Clean the bio-hood with 70% ethanol, both the inside of the glass and work surface.
UV the bio-hood at the end of each day.
18. Repeat steps 1-18 each time the cells meet 50-80% confluency (which can take 2-5
days) for each culture flask until a total of 15 separate culture flasks have been used.

Part E: Applying Chemicals

1.

Label the 24 microplate cover with a permanent marker as shown in figure 4

(labeling rows for every hour checked and
columns for concentration of chemical or control).

2. Using a 500-1000L micropipette, put 750 L of

breast cancer cells with media into every cell of
the microplate, excluding row D and column 6. Figure 4: Diagram of how to label and

distribute chemical concentrations in 24

wellplate.

3. Dispose micropipette tip.

4. Dial down the same micropipette down to 250L and pipette sinigrin hydrate 100M

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into the column 1, excluding cell 1D, taking care not to touch the sides of the plate
cell (if touch, simply dispose and replace micropipette tip). Dispose tip.
5.

With a new tip, micropipette 250L of sinigrin hydrate of the concentration 10M
into column 2, excluding cell 2D, taking care not to touch the sides of the plate cell
(if touch, simply dispose and replace micropipette tip). Dispose tip.

6. Repeat steps 14-15 for allyl isothiocyanate, using cell columns 3 and 4.
7. With a new tip, micropipette 250L distilled water (from erlenmeyer flask) into
column 5, excluding cell 5D. Dispose tip.
8. Place the tray into the CO2 incubator.

Part F: Using the Hemocytometer

1.

24 hours after first mixing chemical solutions with cancer cells, remove the
microplate from the CO2 incubator and place it on the work surface.

2. Clean a hemocytometer by first running it under water and then spraying with 70%
ethanol. Dry first with clean paper towels, and then further dry and clear with lense
paper.
3. Using a 200L micropipette, transfer 100L of cancer cells and
media from cell 1A in the original microplate to cell 1A in a new
24 microplate. Dispose tip.
4. With a new tip, put 100L of trypan blue into cell 1A of the new
plate. Dispose tip.
*Remember: dont let solution sit for more than 5-10 minutes.
5.

Figure 5: Diagram of
one hemocytometer
grid under the
microscope,
highlighting which
grids to count cells in
(MicrobeHunter, 2010).

While gently holding the cover slip down, gently micropipette (with the 2-40L

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micropipette and according tips) 10L of the dyed cells into the side of the coverslip
on the hemocytometer, allowing capillary action to carry the liquid up. Refill and
repeat. (If there are air bubbles, clean as in steps 1-2 and repeat step 5)
6. Turn on and place hemocytometer under 4X microscope magnification. After finding
the top grid set, as shown in figure 5, zoom into 10X magnification.
7. Counter in each hand, designate one hand to dead cells, and another to viable cells.
Starting in grid 1, count the cells dyed blue as dead, and clearish bubbles as live
(shown in figure 6).

Figure 6: Difference between dead and viable cells under

microscope in hemocytometer

8. Repeat step 7 for grid 3, 5, 7, and 9 (as shown in figure 5).

9. Record numbers for viable and dead cells.
10. Move microscope over the bottom grid set and repeat steps 7-9, using the same
designated hands to count dead and alive cells.
11. Repeat steps 1-10 for cells 2A, 3A, 4A and 5A.
12. Repeat steps 1-11 four times, moving down rows (B, C, D) in new microplate to make

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space for dyed cells solution.

13. Replace cell plate in CO2 incubator and clean up materials. To clean the new cell
plate, generously spray with bleach and rinse with water. Clean workspace with 70%
ethanol.
14. Repeat steps 1-13 every 24 hours, pulling cells from the designated row (as shown in
figure 4).

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Variables:
Independent Variables: Cruciferous vegetables (sinigrin & allyl isothiocyanate)
Dependent Variable: cancer rate of apoptosis (as observed with a hemocytometer)
Control: water (3 cells on well plate)
Constants:

Experimenter ensuring consistent methods throughout the experiment,

there was only one person splitting cancer cells, making chemical solutions,
and counting the number of viable and dead cells in the hemocytometer

CO2 incubator the same incubator was set at 36.5 C & 5.0% CO2 for the

duration of the cell growing and splitting period, helping the cells grow at a
similar rate throughout

Plain media media from the same original bottle of DMEM was used for

every cell splitting session

Complete media the mixture of media was mixed using the same DMEM

used as for plain media, insulin, penstrep, and FCS, creating one solution that
fed all the cancer cells throughout experimentation

Culture flask brand all originated from the same company and supply

order, suggesting very similar conditions within the factory that would
similarly affect parts exposed to cancer cells

trypsin same batch ensures that the cells would release from the culture

flasks at roughly the same rate and have the same level of (minimal)
contamination

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Micropipette-tip sets same unused box would hold (more) similar

quantities of fluid and (low) contamination

Micropipettes pull the same amount of fluid (with use of the same volume)

Electric pipetting pump pull fluid at the same rate every time, and with the

same experimenter, the suction button would be held at the same pressure
for the same amount of time

Bio-hood same ideally sanitary conditions & same level of UV radiation in

preparing materials and hood for future use

37 C water bath cancer cells exposed to the same temperature medias and

thus put at risk (or not) at the same level

Hemocytometer the same sizes and number squares with the same

scratches ensure increased accuracy along with the experimenter's familiarity

with which spots are credible data and which are simple mistakes

Microscope same magnifications and lighting conditions for counting cells

Counters require the same amount of pressure to trigger and give the

experimenter a greater sense of familiarity, thus more accurate counts

Trypan blue bottle same source of coloring that effects the cells in the

same way, with the same size penetrating dye particles (smaller would dye
more cells and bigger would dye even fewer cancer cells) and reacts to the
cells in the same way

24 Well-plates exposed to the same amounts of outside of outside factors,

(BPAs, etc.) and same original sanitation level

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Results:
Raw Data: Viable & Dead Cells
Sinigrin Hydrate

100 M

10 M

Allyl Isothiocyanate

100 M

Water (control)

10 M

Viable Dead Viable Dead Viable Dead Viable Dead Viable Dead
7
15
11
14
13
10
24
6
16
4
Trial
1
--------24
8
14
3
30
14
24
hrs Trial
7
6
25
9
11
5
15
2
4
5
2
15
11
24
15
5
4
21
2
3
5
8
15
12
12
2
7
2
2
17
7
Trial
1
24
21
4
4
1
4
3
5
8
4
8
7
3
3
14
9
8
4
4
6
Trial
2
31
19
8
4
10
9
2
4
9
6
48
hrs Trial
6
3
8
6
5
10
10
5
3
0
3
4
7
5
3
1
4
6
5
6
8
16
4
18
4
2
4
8
4
7
4
Trial
4
31
16
13
2
2
7
4
4
16
9
6
11
4
8
81
29
49
22
5
8
Trial
1
5
4
3
6
11
13
6
6
8
7
5
11
9
5
7
6
10
14
4
9
Trial
2
4
4
4
4
4
7
11
18
3
7
72
hrs Trial
0
6
73
22
7
8
8
8
0
3
3
2
6
8
3
8
4
1
0
4
16
33
9
4
7
4
34
3
3
7
5
Trial
4
3
13
15
14
8
32
2
0
1
8
Table 1: Number of viable and dead cells for each set of trials and chemical solution, counted every 24 hours.
Both sets of data for every trial comes from each of the two marked viewing cites in the hemocytometer.

Figure 8: Hemocytometer under microscope at 10X

magnification for trial 3B of sinigrin 100 M

Figure 7: Hemocytometer under microscope at 4X

magnification for trial 3B of sinigrin 100 M

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Stauber
viable cells

Percent Viability (

24 hrs.
48 hrs.
72 hrs.
Avg.

/viable + dead cells )

Sinigrin
100 M

Sinigrin 10
M

AITC 100
M

AITC 10
M

47.73%
56.43%
36.83%
47.00%

59.67%
63.10%
52.19%
58.32%

63.98%
34.31%
44.19%
47.49%

85.40%
55.99%
62.33%
67.91%

Water
(control)
57.50%
63.50%
30.25%
50.42%

Table 2: Average percent of viable cells as measured by number of live cells out of total
cells counted per time trial every 24 hours Percent expected to decrease from first trial
time at 24 hours to 72: as time goes on, the cells age and die (especially without media
replacement). Viabilities of higher molarities should also average over the the three test
times as lower than their 10 M counterparts.
100.00%
90.00%

Percent Viability

80.00%
70.00%
60.00%

Sinigrin 100 M
Sinigrin 10 M
AITC 100 M
AITC 10 M
Water (control)

50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
24

48

72

Time (hours)
Figure 9: Graph of average percent viabilities through time

Aver ag e P er cent Viability

80.00%
70.00%

Percent

60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%

Sinigrin 100 M

Sinigrin 10 M

AITC 100 M

AITC 10 M

Water (control)

Chemical
Figure 10: Total average percentage of viable cells for each molarity of chemical & control

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Stauber

Cell Concentration (

viable cells

/mL )

avg. # cells per sq. x dilution factor 104

24 hrs.
48 hrs.
72 hrs.
Avg.

Sinigrin 100
M
386666.67
640000
290000
438888.89

Sinigrin 10
M
800000
355000
650000
601666.67

AITC 100 M
630000
185000
650000
488333.33

AITC 10 M
740000
217500
450000
469166.67

Water
(control)
380000
350000
520000
416666.67

Table 3: Average calculated number of cells for every mL of mixed chemical, media, and cell
solution. Expected to be very similar through all the trials, considering cells come from
same source that grew in the same media (only differences due to growing in separate
culture flasks).

Cocentration (viable cells / mL)

1000000
900000
800000
700000
600000

Sinigrin 100 M
Sinigrin 10 M
AITC 100 M
AITC 10 M
Water (control)

500000
400000
300000
200000
100000
0
24 hrs.

48 hrs.

72 hrs.

Time (hours)

Cocentration (viable cells / mL)

Figure 11: Graph of average calculated number of cells per mL of chemical, cell, and media solution

A v er a g e Cel l Co nc ent r a t io n
1000000
800000
600000
400000
200000
0
Sinigrin 100 M

Sinigrin 10 M

AITC 100 M

AITC 10 M

Time (hours)

Figure 12: Graph of total average cell concentration per chemical solution

26

Water (control)

Stauber

Statistical Analysis:
Statistical Significance of Percent Viability (P-Value)
Sinigrin 100 Sinigrin 10
M > AITC M > AITC 10 Sinigrin 100 Sinigrin 10 AITC 100 M AITC 10 M
100 M
M
M < Water M < Water
< Water
< Water
24 hrs.
0.938
0.989
0.252
0.559
0.711
0.982
48 hrs.
0.0054
0.149
0.2097
0.481
0.00177
0.173
72 hrs.
0.734
0.818
0.718
0.982
0.9
0.993
Avg.
0.5591
0.652
0.3932
0.674
0.5376
0.716
Table 4: Statistical significance of the comparison between percent viabilities with chemicals of different
molarities and water. With an -level of 0.95, any p-Value lower than 0.05 is deemed as significant and
thus proves the hypothesis of comparison.

1
0.9
0.8

P-Value

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
24 hrs.

48 hrs.

72 hrs.

Time (hours)

Sinigrin 100 M >

AITC 100 M
Sinigrin 10 M >
AITC 10 M
Sinigrin 100 M <
Water
Sinigrin 10 M <
Water
AITC 100 M <
Water
AITC 10 M <
Water

Figure 13: Graph comparing P-values (statistical significance) at each time marker

A v er a g e S t a t is t ic a l S ig nific a n c e
1

P-Value

0.8
0.6
0.4
0.2
0

Sinigrin 100 M > AITC 100 M

Sinigrin 100 M < Water

AITC 100 M < Water

Comparison
Figure 14: Graph comparing average statistical significance (P-value) of each chemical-chemical
comparison at each time marker

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Stauber

Taking into account the remarkably high p-Values (which measure correlation for the
purpose of proving a hypothesis, taking anything under 0.05 as a good correlation) across
the charts, it is difficult to say that there is any specific correlation. While there are two
separate instances that appear to provide evidence that 100 M allyl isothiocyanate is
indeed the better cancer killer, the rest of the p-values almost entirely point in the total
opposite direction. Thus, as stated above, it is again shown that there is a great likelihood
that glucosinolate sinigrin and allyl isothiocyanate work through bodily functions. To prove
anything for certain, though more trials on a greater magnitude would be needed. In other
words, the findings are ultimately inconclusive.

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Stauber

Experimental Error:
In the duration of the whole experiment, there are a great many places where one
can make an error that can potentially effect the entire project.
While culturing and preparing cells for storage, one can kill the cells by simply not
warming the media or trypsin up to the needed temperature of about 37 C. A similar fate
can also come if the trypsin is left on the cells for too long. The way trypsin works is by
breaking bonds between the cells and the plastic of the culture flask, but that same action
can quickly turn and destroy cells. Another issue that may arise is if the complete media is
not made properly, thus slowing the replication or altogether killing the cells. Upon
applying chemicals and adding media, there is also a contamination factor that comes into
play: if the cells are contaminated by something harmful, the cancer cells may again, die
and/or no longer be viable for testing. While cleaning with ethanol before and after
coming in contact with cells and working under a bio-hood lower chances for
contamination, if the cells are opened and exposed to contaminated air or someone's
sneeze, they can be prone to damaging or DNA-changing bacteria or viruses (which would
require restarting cell-splitting.
In creating the chemical solutions, there are many possible problems arise, again
many of them pertaining to contamination. By not switching micropipette tips between
use, or even using the same water for each flask mix, other outside factors may come into
play in regards to testing the cells. Also, not having Erlenmeyer flasks that have been
thoroughly cleaned may likewise lead to contamination or reaction between chemicals
added and chemicals that had previously been in those flasks. Labeling wrong and/ or

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accidentally adding the wrong amount of water may also create different results than those
expected or desired later down the road.
The easiest and most noticeable mistakes, however, regard counting cells with the
hemocytometer. Especially for first-time users, cells may be incorrectly identified, and with
small trials, this can significantly skew data. This may be accentuated by having leftover
particles on the hemocytometer, so when the cells with trypan blue dye are added, the
particles may be dyed blue to appear like dead cells, and if not known or noticed, this can
significantly skew data for all trials. If the hemocytometer is not washed properly, not only
can there be extra particles remaining, but also contamination of samples from previous
trials. Something else that could give inaccurate data is either counting too many or too
few cells by counting in the wrong quadrants. A small, unlikely error may also occur if the
counters are not only clicked when viewing a cell (such as sneezing or hand spasms).
In the end, there are many possible errors, some of which likely occurred in this
experiment.

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Conclusion:
From the data collected, it can be concluded that neither hypothesis (regarding
chemical induction of apoptosis, or AITC being the superior inducer) can be significantly
refuted. While some data points suggest that there is indeed a correlation between higher
concentration (100 M) chemicals and higher die-off rates, it is not significantly higher in
comparison to the control. The data also shows that the low 10 M of solution seem to
actually feed the cancer cells (as compared to the lowered percent viability from the
control).
In contradiction to the first of the original hypotheses (regarding direct induction of
apoptosis), the control had nearly a 10% higher die-off rate than the sinigrin 10 M solution
and 20% than than AITC 10 M solution. With chemicals known to prevent and slow cancer,
neither low concentration was proven more effective than distilled water. But on the other
hand, both 100 M solutions were found to have about 3% higher potencies (as shown in
table 2) than the control, thus contradicting previous findings (in lower concentrations) and
providing some evidence to prove that both higher concentrations allyl isothiocyanate and
sinigrin are inducers of apoptosis of mammary carcinoma. However, considering the
proximity between the average viability percentages and very high p-values (as shown in
figure 13), this information should not be taken as definite proof that either allyl
isothiocyanate nor sinigrin are direct inducers of apoptosis (meaning that they do not work
through bodily systems like the immune system).
Additionally, it appears that the data points of the higher concentration of sinigrin
hydrate is a stronger inducer of apoptosis (as compared to allyl isothiocyanate and water),

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furthermore refuting my hypotheses. While nevertheless insignificant, the data still points
to sinigrin (having a significantly higher p-values than 0.05, thus deeming it insignificant).
Ultimately, taking all the information into account, the best conclusion drawn is that
more trials are necessary to truly refute or support either hypotheses. If similar trends
occur in the next set of trials, one may deem it safe to say that neither allyl isothiocyanate
nor sinigrin induce mammary carcinoma apoptosis through direct contact. In that case, the
best way to discover exactly how these chemicals interact with breast cancer would be to
observe them in vivo, or how they would interact naturally within the human body. (Doing
small tests directly on cancer cells only sees one aspect of a whole system.) With new
technology, this may be able to go beyond a simple correlational study between chemical
intake and chances of developing such cancer and rather utilize nanobots for observing the
interaction.
From the inconclusive data collected, it is best implied that the most effective way to
utilize cruciferous vegetables in fighting breast cancer may be simply eating such
vegetables as broccoli, Brussels sprouts, and cauliflower, but more testing is necessary to
show if true or not. Considering the slight evidence that sinigrin may be more effective, it
may be best to cook the vegetables just to the point where the catalyst myrosinase is
deactivated, so the sinigrin remains a glucosinolate when consumed.

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