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ABSTRACT
HeLa cells are a type of cancer cell lines and are immortal. All HeLa cells are derived from a
woman who had cancer in the cervix. This project was done to see response of HeLa cells against fresh
extract of garlic, ginger and onion. It was hypothesized that these compounds would inhibit or slow down
the growth of the HeLa cells. Research done prior to the experiment suggests that these compounds may
inhibit the HeLa cells growth. To experiment on this project, extracts of garlic, ginger, and onion were
added in 12-well tissue culture plates containing HeLa cells. They were incubated overnight in the tissue
culture incubator. The next day MTT was added to the cells. After the MTT was added, the plates were
incubated back at 370 C cell incubator for 3-4 hrs or until the purple product was formed (MTT is a
substrate for mitochondrial enzyme in live cells) in the wells containing live HeLa cells. The purple
product was dissolved in SDS solution. The solutions in each well were placed in cuvettes and put into the
spectro- photometer. The intensity of the purple color was recorded by reading the absorbencies of the
purple solution at 570 nm. Also the extracts treated and untreated cells were checked under the
microscope and photographs were taken.
The results show that garlic, ginger, and onion all slowed the HeLa cell growth. The average
spectrophotometer readings (absorbencies) for filtered 100ul garlic, ginger, onion, positive control (HeLa
cell not treated with extract), and negative control (medium only) were, 0.208, 0.699, 0.700, 1.102, 0.000,
respectively. The average readings for 100ul non-filtered garlic, ginger, onion, positive control (no
extracts), and negative control (medium only) were 0.279, 0.177, 0.860, 1.102, and 0.000, respectively.
This gives us the percent growth inhibition of HeLa cells incubated with non-filtered extracts of onion,
garlic, and ginger as follows: 22%, 84%, and 75%, respectively. For the filtered extracts of onion, garlic,
and ginger the values are: 36%, 81%, and 37%, respectively.
This research study is unique to show the inhibition of HeLa cell growth by the herbal compounds
mentioned above. The results obtained from my project suggest that use of fresh garlic, ginger, and onion
may help in fighting cancer.

Key words: MTT [3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide] is a

reagent used to determine the cell viability.
SDS, Sodium Dodecyl Sulfate

INTRODUCTION
The cell is the unit of structure that all plants and animals are made up of. The cell is the smallest
unit in the living organism that is capable of integrating the essential life processes. There are many
different types of cells and each has a specific function. Cancer begins when one cell starts growing and
dividing out of control. This cell divides into a mass of cells called a tumor.
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The oldest description of human cancer was found in an Egyptian papyri written between 30001500 BC. It referred to tumors of the breast. The oldest specimen of a human cancer was found in the
remains of a female skull dating back to the Bronze Age (1900-1600 BC). The mummified skeletal
remains of Peruvian Incas, dating back 2400 years ago, contained lesions suggestive of malignant
melanoma. Cancer was found in fossilized bones and manuscripts of ancient Egypt. This shows that
cancer is not just a recent disease.
Benign tumors involve cells that stay in the same place and stop growing before it gets very large.
Malignant tumors (more commonly known as cancer) are tumors in which the uncontrolled dividing cells
invade and destroy healthy tissues elsewhere in the body. Metastasis is the spread of cancer cells beyond
their original site. When metastasis occurs, the cancer cells break away form the malignant tumor and
travel to other parts of the body, where they invade healthy tissue and begin forming new tumors. There
are different types of cancer according to types of tissues they effect.

Carcinomas- grow in the skin and the tissues that line the organs of the body. Examples of carcinomas
are breast cancer and lung cancer.
Lymphomas- solid tumors that grow in the tissues that form blood cells. An example is leukemia
(uncontrolled production of white blood cells).
Sarcomas- grow in bone and muscle tissue.

Lung cancer is the leading cancer killer in both men and women. Smoking is the number one cause
of lung cancer. Cigarette smoke contains more than 4,000 different chemicals, many of which are proven
carcinogens. The more time and quantity you smoke, the greater your risk of lung cancer. But if you stop
smoking, the risk of lung cancer decreases each year as normal cells replace abnormal cells. After ten
years, the risk drops to a level that is one-third to one-half of the risk for people who continue to smoke. In
addition, quitting smoking greatly reduces the risk of developing other smoking-related diseases, such as
heart disease, stroke, emphysema and chronic bronchitis. There are two types of lung cancer: small-cell
lung cancer and non-small-cell lung cancer.
Breast cancer is the most common cancer among women, excluding non-melanoma skin cancer.
Currently, approximately 3 million women in the US are living with the disease, including 2 million who
have already been diagnosed, and another 1 million who do not yet know they have the disease.

Leukemia is cancer in the bone marrow in which the malignant cells are the white blood cells
(leukocytes). Leukemia cells begin to multiply in the marrow and they crowd out the normal blood cells
that carry oxygen to the body's tissues, fight infections, and help wounds heal by clotting the blood.
Leukemia can also spread from the marrow to other parts of the body, including the lymph nodes, brain,
liver, and spleen.
There are many different causes of cancer. Mutations that alter the expression of genes coding for
growth factor proteins can lead to cancer. There are certain genes in the human body called protooncogenes. Proto-oncogenes code for proteins that regulate the rate of the cell cycle by controlling cell
growth, cell division, and the ability of cells to stick to other cells. An oncogene is a gene that causes
cancer. Oncogenes begin as proto-oncogenes. A mutation in a proto-oncogene may cause it to produce
more protein or a protein that seems to be unusually active in triggering cell division. Tumor-suppressor
genes are genes that code for proteins that prevent the uncontrolled rate of cell division. When these genes
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mutate, the proteins that they code for are defective, thus causing a predisposition to cancer.
These mutations can occur anytime, but are more likely to appear if the organism has had exposure
to carcinogens. A carcinogen is a substance that increases the risk of cancer (tobacco, asbestos, and
ionizing radiation, such as X rays or ultraviolet light from the sun). Most carcinogens are mutagens
(substances that cause mutations within the cell). Certain viruses can cause cancer. Their genes are
actually onco-genes. Viruses also stimulate uncontrolled growth in host cells by causing mutations that
accelerate the rate of cell division.
In normal cells, the frequency of cell division is governed by several factors. Normal cells will
stop dividing when they become too crowded, usually after 20 to 50 cell divisions. Cancer cells, however,
continue dividing after they are no longer attached to other cells, a trait that facilitates the spread of cancer
cells throughout the body.
Whether a person develops cancer depends on many factors. Some families may have a higher risk
of a certain type of cancer due to genetic make-up. Also, the amount and number of exposures to a
carcinogen plays a role. Usually, though, more than one mutation is needed for an organism to develop
cancer. This explains why people with older age are more likely to develop cancer. The longer they live,
the more mutations they will probably accumulate.
Cancer cells are cells that do not respond to signals that inhibit growth. Normal cells pass through
a limited number of cell divisions (50 is about the limit) before they decline in vigor and die. This may be
caused by their inability to synthesize telomerase. Cancer cells may be immortal; that is, proliferate
indefinitely in culture. For example, HeLa cells are cultured in laboratories around the world. They are all
descended from cells removed from a cancer (of the cervix) of a woman who died of her disease almost a
half century ago. Cancer cells in culture produce telomerase. Normal cells, when placed on a tissue culture
dish, they proliferate until the surface of the dish is covered by a single layer of cells just touching each
other. Then mitosis ceases. This phenomenon is called contact inhibition.
Normal cells go through a finite number of cell divisions before they die. This is caused by their
inability to produce telomerase. Cancer cells in culture produce telomerase, an enzyme which allows them
to grow and survive indefinitely. In theory, a lack of telomerase slow the growth of tumors by causing the
cancer cells to concede before they did much damage.
The common cancers are not curable in an advanced stage, and a large number of patients with
metastatic disease who have no curative treatment options are searching for therapy. Metastatic cancer
patients are a vulnerable population willing to use nontraditional methods of treatment in spite of limited
knowledge.
There have been many studies conducted involving herbal compounds and cancer. There was a
study conducted in Shanghai, China where researchers compared the diets of individuals with early or
advanced prostate cancer to the diets of healthy individuals. In the study, individuals who reported eating
the most allium vegetables (garlic, scallions, onions, chives, and leeks) were found to have a nearly 50%
lower cancer risk than those who ate the least.

PURPOSE
HeLa cells are the first continuously cultured human malignant cell line derived from the cervix of a
woman. My question is, "Does garlic, ginger, and onion inhibit the growth of HeLa cells?"

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MATERIALS
Frozen stock of HeLa cells, T-75 flasks, incubator, DMEM HeLa cell medium, high-power microscope,
sterile hood, 70% ethanol, aspiration vacuum, PBS (phosphate buffer saline), trypsin EDTA,
hemocytometer, 12-well and 6-well cell culture plates, fresh ginger, fresh garlic, fresh onion, Pipetman,
tips, 25-micron cheesecloth, falcon tubes, syringe, 0.45 and 0.2-micron filters, MTT powder, 10%
SDS(Sodium Dodecyl Sulfate)-solution, DMSO (Dimethyl Sulfoxide), shaker, centrifuge machine,
Pasteur pipettes, aluminum foil, spectrophotometer, and spectrophotometer cuvettes.

METHODS
Growing HeLa cells
1) Take out frozen stock of HeLa cells
2) Thaw at 37o and add 1.0 ml of HeLa cells to 12.0 ml of DMEM medium in T-75 flask.
3) Put flask in 37o incubator and wait until they are 100% confluent (approx. 2 days)
Filtering
1) Have garlic, ginger, and onion juice ready by filtering through a 25-micron cheesecloth.
2) Under a hood, take a syringe and suck up the garlic juice. Apply a 0.45-micron filter and push contents
into another tube.
Splitting cells
1)
2)
3)
4)
5)
6)
7)

Take out flask and check under microscope to see if cells are confluent.
Take flask to hood, and clean workstation with 70% ethanol.
Aspirate the medium with a vacuum.
Wash the cells 3 times with PBS (phosphate buffer saline)
Add 1 ml of trypsin-EDTA to the flask.
Add 12ml of the medium to the flask to stop the trypsin effect.
Count cells with hemacytometer and cells were ready for further use.
Experimental Design

8) Add 1ml of DMEM medium in each well for the 12 well plates (2 plates), plate #1 and #2. Add 0.25
ml of HeLa cells (that were just split) 2nd, 3rd, and 4th columns of each plate.
9) Incubate the plates for 2 hours in 37o incubator.
10) Take out both of the plates and add onion, ginger, and garlic extracts as written in the plates.
Plate # 1
Medium only (0.3ml)

Cells (0.25 ml) only + 50

ul PBS

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Cells (0.25 ml) + 50ul

non-filtered onion

Cells (0.25 ml)+ 100ul

non-filtered onion
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Medium only (0.3ml)

Cells (0.25 ml) only + 50

ul PBS

Cells (0.25ml)
+ 50ul filtered onion

Cells (0.25ml) + 100ul

filtered onion

Medium only (0.3ml)

Cells (0.25 ml) only + 50

ul PBS

Cells (0.25ml) +50ul

non-filtered ginger

Cells (0.25ml) + 100ul

non-filtered ginger

Plate # 2
Medium only
Cells (0.25ml)
Cells (0.25ml) +50ul
(0.3ml)
+100 ul PBS
filtered ginger
Medium only
Cells (0.25ml)
Cells (0.25ml) +50ul non(0.3ml)
+100 ul PBS
filtered garlic
Medium only
Cells (0.25ml)
Cells (0.25ml) + 50ul non(0.3ml)
+100 ul PBS
filtered garlic
(the 50 & 100ul PBS was added to the control wells to keep the same volume)

Cells(0.25ml) + 100 ul
filtered ginger
Cells (0.25ml) + 100ul
non-filtered garlic
Cells (0.25ml) + 100ul
non-filtered garlic

11) Keep plates in a 37o degree incubator and let it grow overnight
Next Day: MTT Assay in the Experiment
1)
2)
3)
4)
5)

Take 30 mg of MTT powder, dissolve it in 6 ml of PBS (5.0 mg/ml), and then filter it.
Take out the plates and put 100 ul of MTT in each well for both plates.
Put plates back in incubator and watch for purple color to develop, approx. ~2 hours.
Take plates out again and put 1ml of 10% SDS solution in each well.
Cover the plates with aluminum foil and put on a shaker (low speed) for ~4 hours. This helps dissolve
the purple product.
6) Next, transfer the liquid of each well in small tubes with the help of a pipette. Keep them in a
centrifuge machine and spin at 1000 rpm (revolutions per minute) for 5 minutes.
7) After done, take tubes out and transfer 0.9 ml into cuvettes with Pasteur pipettes.
8) Place the medium only cuvette in the spectrophotometer and set it to zero (This is blank reading). Read
the absorbance at 570 nm. One by one, read the rest of the tubes in the spectrophotometer and record
their readings.
9) After I started getting consistent results, the experiment was repeated 4 times. Graphs of the results
obtained, from the average data, are presented in the RESULTS section.
------------------------------------------------------------------------------------------------Standardization of the MTT Assay
NOTE: This procedure was done to find if SDS or DMSO worked better to solubilize the purple product
and also to check the validation of the method by increasing the number of cells. I found that SDS works
better.
Set up was as follows: This was done in two, 6 well plates.
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2.0 ml of medium + 0.5 ml of cells

2.1 ml of medium + 0.4 ml of cells

2.3 ml of medium + 0.2 ml of cells

2.4 ml of medium + 0.1 ml of cells

Nothing

Nothing

2.2 ml of medium +
0.3 ml of cells
2.5 ml of medium +
0.0 ml of cells
Nothing

Keep plates in a 370 C incubator and let it grow overnight. Rest of the procedure was same as in the
Next Day MTT Assay. The only difference was that in one plate SDS was added and in other DMSO
solution.
The data obtained was linear with both but SDS was dissolving the product faster. The reliability of the
method is shown in the result section.

RESULTS
Therefore the assay was used to determine the cell viability under different treatment conditions along
with non-treated (pos. control)

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GRAPHS for the actual experimental data

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Microscopic photographs of HeLa Cells: Note untreated con

crystals which indicates live cells.

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DISCUSSION
This project was
undertaken with the idea to see the
effect of herbal products like
garlic, ginger, and onion on the growth of cancer cells (HeLa cells). My results show that the fresh
extracts of garlic, ginger, and onion have the ability to slow the HeLa cell growth to a different degree.
For the non-filtered extracts, the growth inhibition for onion, garlic, and ginger was 22%, 84%,
and 75%, respectively.
For the filtered extracts, the growth inhibition for onion, garlic, and ginger was 36%, 81%, and
37%, respectively.
Garlic is most inhibitory compared to the other 2 compounds in both filtered and non-filtered
extract experiments. I was surprised to see a very significant difference between filtered and non-filtered
ginger. Non-filtered ginger is inhibiting the HeLa cell growth to almost the same extent as garlic. These
results made me think that a compound in ginger which can not be filtered through 1.2 micron, is quite
active in killing the HeLa cells. Onion was the least effective, but still had a little effect.
Many herbal compounds have been shown and discussed to have an effect on cancer. There are
some compounds in garlic which may have anti-cancerous properties. The allyl-sulfur compounds are the
main ones. These compounds are reported to prohibit cancer by decreasing the activation of carcinogens
in the body, reducing the production of carcinogens, and increasing the body's ability to repair damaged
DNA. Although there have been no human studies on ginger's ability to stop tumor growth, there have
been few experiments done on animals. It has been said that ginger reduces nausea and vomiting during
chemotherapy.
These compounds work against HeLa cells, which is very interesting and exciting. This research
adds up to the knowledge of Herbs and Cancer treatment or Prevention. I should extend this project
by testing different cell lines with these compounds, including primary cell lines. I should also try to
identify the compound in non-filtered ginger extract which is probably involved in killing HeLa cells.
The evidence is shaping up that allium vegetables are an important component of a cancer-protective diet
high in plant foods.

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CONCLUSION
This project showed the effect of garlic, ginger, and onion extracts on the growth of HeLa cells. Garlic,
ginger, and onion all slowed the growth of the cells. Garlic did the best overall. From this project, it can be
concluded that garlic, ginger, and onion are effective in slowing the growth of HeLa cells. This project
also shows how herbal remedies can be used as complimentary alternatives in preventing cancer.

EXPANSION
1) Effect of garlic, onion, and ginger on other cell lines (ex. primary cell lines)
2) Effect of other compounds on HeLa cells (ex. bitter melon and turmeric)
3) Effect of different forms of garlic on HeLa cells or other cell lines (ex. garlic powder and aged garlic
extract.)

ACKNOWLEDGEMENTS
I would like to thank Dr. Gillian Air (Dept. of Biochemistry, OUHSC) and her lab colleagues in letting me
use their lab and equipment and for aiding me in my experiment.

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REFERENCES
1. Almco, Edward., Bogin, Barry., Chubb, Curtis., Ehmann, William., Feil, Mark., Hershey, David.,
Leslie, Mitchell., Liem, Karel., Thwaites, William., Tocci, Salvatore. (1999). Modern Biology. Austin,
Texas: Holt, Rinehart and Winston
2. Franks, L. M., Teich, N.M. (1997). Introduction to the Cellular and Molecular Biology of Cancer. New
York: Oxford University Press Inc.
3. American Institute for Cancer Research, Charity Wire. (2002). Cancer Experts Encouraged by New
Garlic/Onion Study. [Online] Available: http://www.charitywire.com/charity10/print_04613.html
4. Scientific American, Greider, Carol W., Blackburn, Elizabeth H. (1996). Telomeres, Teolomerase and
Cancer. [Online] Available: http://www.genethik.de/telomerase.htm
5. American Institute for Cancer Research. (2003). Cancer Basics. [Online] Available:
http://www.aicr.org.uk/CancerBasics.stm
6. Banerjee SK, Mukherjee PK, Maulik SK. (2003). Garlic as an antioxidant: the good, the bad and the
ugly. [Online] Available: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?
cmd=Retrieve&db=PubMed&list_uids=12601669&dopt=Abstract
7. American Cancer Society. (2000). Complementary and Alternative Cancer Methods. USA: American
Cancer Society.

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