Calli Walsh

Calli Walsh

University of Wisconsin – Stout

Calli Walsh

walshc@my.uwstout.edu

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Calli Walsh

Introduction

According to the American Cancer Society, this year alone in the United States, 1,596,670 new

cases of cancer are expected to be diagnosed, with 571,950 of those cases ending in death

(American Cancer Society, 2011). Today, cancer is one of the most common causes of death,

exceeded only by heart diseases. Because there are so many variations of cancer, the disease

has become extremely complex and difficult to treat, let alone cure. However, doctors and

scientists are hard at work researching ways to battle the disease, sometimes incorporating

unthought-of allies such as plants.

A promising drug known as Taxol (paclitacxel), which is isolated from the bark of Pacific Yew

trees, is currently being used to suppress breast cancer (Pandi. M., 2010). Extracts made from

pomegranates have proved to inhibit prostate cancer cell growth (Adhami, VM., 2012), an

advancement for patients at high risk of developing prostate cancer. Promising drugs like these

are the result of research on unknown plant species in attempts to better understand their

medicinal properties. It has been said that plants are a main source of biologically active

chemicals that can help prevent, fight, and cure diseases. Despite this fact, only 10 % of all the

plants on Earth have actually been studied, meaning more research is needed (Talib, Wamidh.

2010).

Tradescantia zebrina (Tz) is a plant species that has remained researched, despite its variety

of medicinal purposes worldwide. Tz leaves are commonly a deep purple color on top with a

silver strip going down the middle and a dark green bottom. Originally found in regions of

Mexico, this plant has been used to make an herbal tea remedy known as matali, and is

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consumed cold as a tonic for improved health. In Jamaica, it is also made into an herbal tea that

helps treat the common cold. In the Hmong culture, it is used as a poultice, to draw out the

infections, to help treat skin wounds, rashes and growths, even though the plant is known to

sometimes cause skin irritation. Medicinal properties such as these suggest the plant may have

anti-cancer properties that could aid in the fight against cancerous diseases.

Tz’s historical uses to treat skin conditions along with its many other medicinal uses

suggest that Tz Extracts may express anti-cancerous properties that could possibly inhibit the

growth of certain cancer cells.

Methodologies

To test this hypothesis, a cancer cell line, A-549 lung carcinoma cells, was treated with

both and aqueous and methanol TZ extract, dissolved in both DMSO and sterile H2O, and cell

numbers were counted 3 separate times over a five day period to determine whether or not

there is any growth inhibition. A control test was also performed on each of the different cell

lines, in which the cells were treated with sterile water instead of extract, allowing

uninterrupted growth. To test whether or not the Tz extracts have any anti-cancer properties,

the treatment was applied to specific lines of cancer cells. Preliminary tests examine the effect

of the extract on the cell growth of facial epidermis cancer cells known as “SCC-13y”s. In this

particular study, a cancer cell line of A-549’s was used to test growth changes as well as to

compare their results to a non-cancerous, “normal” cell line of HFF1 fibroblasts. Two methods

of extraction, water and methanol, were also compared in an attempt to isolate the active

compounds from the extract.

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A-549 cancer cells, also known as human lung adenocarcinoma epithelial cells, were

derived from a fifty-eight year old Caucasian male and will be provided by American Type

Culture Collection, or ATCC.org. SCC-13y cancer cells, also called squamous cell carcinoma cells,

were derived from a fifty-six year old woman and will be provided by ATCC.org as well. The HFF-

1 cells, which are human foreskin fibroblasts, are a non-cancerous cell line derived from two

newborn males ATCC provided as well.

All cells were grown in 100cm2 culture dishes and incubated in a NAPCO Series 8000WJ Water

Jacketed CO2 incubator at 37OC at 5% CO2. Cells were passed weekly, or when they reached

maximum confluence, using standard trypsin protocols.

Materials and Reagents

DMEM media (Dulbecco’s Modified Eagle Media) was supplemented with 10% Fetal Bovine

Serum and 1% 1x Penicillin Streptomycin was applied to the cells on a regular basis to ensure

cell growth while being grown in an environment continuously kept at 37OC at 5% CO2. Trypsin

(10mg), 10x PBS, sterile H2O, DMSO, 60 cm2 and 100 cm2 culture dishes, and trypan blue were

used.

Tradescantia zebrina plants were provided by and kept in the green house facility at the

University of Wisconsin – Stout where they were cared for under standard conditions.

Aqueous Extract

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A 10 g/mL aqueous extracts was made by mixing 5 grams of Tradescantia zebrina plant leaf

material, ground up with a mortar and pestle, with 50 mL of sterile water. This mixture was

then placed in a 100OC water bath for approximately 15 minutes before it cooled and

centrifuged for 8 minutes. Afterwards, 1 mL of the crude extract was transferred into 30 (or less

depending on need) weighed microfuge tubes and then dehydrated in a lyophilizer for 9 hours.

Once dry, each tube was re-weighed and the total dry weight of the extracted material was

determined. To make a 10 g/mL solution, a calculated amount of sterile water was added to the

first tube in order to dissolve the pellet. Once all particles were settled and the pellet was

dissolved as much as possible, the liquid was transferred into the second tube. This was

continued until all pellets in all the tubes had been dissolved. Once the last pellet was dissolved,

the liquid was transferred into a 15 mL conical tube and the steps were repeated, next time

with DMSO. Finally, each extract (both DMSO and sterile water) was filter sterilized with a 0.22

micron syringe filter to ensure sterility. Both extracts were labeled and stored at 4OC in the labs

refrigerator and kept continuously cold. Before it was applied to cells, it was warmed in a water

bath to 37OC.

Methanol Extract

A 10 g/mL methanol extract was made by mixing 5 grams of Tradescantia zebrina plant leaf

material, ground up with a mortar and pestle, with 50 mL of methanol. This mixture was placed

in a 100OC water bath for approximately 15 minutes before it cooled and was centrifuged for 8

minutes. Afterwards, 1 mL of the crude extract was transferred into 30 (or less depending on

need) weighed microfuge tubes and then dehydrated in a lyophilizer for 9 hours. Once dry,

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each tube was re-weighed and the total dry weight of the extracted material was determined.

To make a 10 g/mL solution, a calculated amount of sterile water was added to the first tube in

order to dissolve the pellet. Once all particles were settled and the pellet has dissolved as much

as possible, the liquid was transferred into the second tube. This was continued until all pellets

in all the tubes were dissolved. Once the last pellet was dissolved, the liquid was transferred

into a 15 mL conical tube and the steps were repeated, this time with DMSO. Finally, each

extract (both DMSO and sterile water) was filter sterilized with a 0.22 micron syringe filter to

ensure sterility. Both extracts were labeled and stored at 4OC in the labs refrigerator and kept

continuously cold. Before it was applied to cells, it was warmed up in a water bath to 37OC.

Growth Curve Analysis

After counting cells with a hemacytometer, 75,000 A-549 cells were plated into fifteen 60cm2

culture dishes and given 3 mL of media each before all plates were incubated at 37OC at 5% CO2.

After 24 hours, the first three plates were counted with a hemacytometer to ensure growth and

to verify the amount of cells seeded. After being counted, 3 µL of the methanol Tz extract

(either DMSO or H2O) was applied to the six treatment plates, while equal concentrations of

sterile water were added to the remaining six negative controls plates. 48 hours later, three

control and three treatment plates were then harvested, using trypsin, and then counted. The

remaining six plates had their media changed and the treatment was re-applied. After another

48 hours of incubation, the final six plates were counted and the cells were disposed of. This

process is simply laid out in Table 1.

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Growth curve analysis graphs were created in Excel. To determine the cell numbers for each

day in both the treatment and control groups, the three counted cell numbers were averaged

and used as plot points.

This same experiment was performed using the SCC-13y cancer cell lines and the data

received was compared to the data from the A-549 experiments.

Day 0 Day 1 (24 hours) Day 3 (72 hours) Day 5 (120 hours)

Control Plates(Sterile H2O)

Seed 9 plates w/ 75,000 cells.

Count 3 plates

*Apply 3 µL of sterile water *

Count 3 plates

*Change media / reapply control*

Count 3 plates

Treatment Plates(Tz extract)

Seed 6 plates w/ 75,000 cells.

* Apply 3 µL of Tz extract *

Count 3 plates

*Change media / reapply control*

Count 3 plates

Table 1: (Time Table for Growth Curve Analsis Data Collection)

Results

One of the most important and time consuming aspects of this research was the need for

consistency when making the Tz extract. Because consistent and precise data was required,

each batch of extract needed to be made at similar concentrations so that the experiments

would result in accurate and reproducible results. Though the most important process, it was

also one of the most difficult to standardize. There are pros and cons to using either an aqueous

extract or the methanol extracts. One of the benefits of using a methanol extract is that it

preserves water soluble molecules that may be present in the extract, which could possibly play

an important role in the turnout of the experiment. For this reason, it was decided that the

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majority of our experiments would be done with a methanol extract, where the pellet

(remaining leaf material) was allowed to dissolve in water and then DMSO, resulting in two

extracts.

Ultimately though, the main problem came from the difficulty of being able to accurately

measure the true amount of extract being produced while also ensuring that it was sterile. After

careful and thorough research, a specific method was created that would help to consistently

reproduce the Tz extract (see Appendix I & II). This method ensured a quantifiable, gram per mL

(weight / volume) extract.

In terms of sterility, numerous steps were taken to ensure that the extract was free of

contaminants and bacteria. In previous tests where the cells had been treated with unfiltered

Tz extract, the plates became contaminated, making it almost impossible to obtain any results.

In an effort to ensure sterility, the first step was to place the crude extract in a 100OC water

bath for fifteen minutes in an attempt to destroy some of the contaminants with heat. Another

important step was the use of a 0.22-µm filter, which was used to filter out unnecessary bits of

plant extracts that still may have been present. When performing experiments with extract that

had undergone these steps, cell cultures grew free of contamination. These results are

represented below in Figures 2 and 3.

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0 1 2 3 4 5 60

200,000400,000600,000800,000

1,000,0001,200,0001,400,0001,600,0001,800,0002,000,000

Control Cells

Treatment Cells

Days

Cell

Num

bers

FIGURE 2: A) (FIRST TEST) GROWTH CURVE ANALYSIS OF DMSO TZ EXTRACTS ON A-549 CELLS, STARTING WITH 30,000 CELLS. B) (SECOND TEST) GROWTH CURVE ANALYSIS OF DMSO TZ EXTRACTS ON A-549 CELLS, STARTING WITH 50,000 CELLS.

0 1 2 3 4 5 60.00

200,000.00

400,000.00

600,000.00

800,000.00

1,000,000.00

1,200,000.00

1,400,000.00

1,600,000.00

Control Cells

Treatment Cells

Days

Cell

Num

bers

Figures 2 and 3 illustrate that Tz extracts have an inhibitory effect on A-549 cell proliferation.

When comparing the growth analysis curves of the treated cells to the control cells, there is a

significant difference between the two. Around 96 hours (4 days) after the treatment or control

is applied to the cells, there is at least a half a million cell difference. Cells treated with either of

the Tz extracts seemed to continued proliferating, but at a much slower rate than the normal

cells (negative control).

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0 1 2 3 4 5 60

200,000400,000600,000800,000

1,000,0001,200,0001,400,0001,600,000

Control Cells

Treatment Cells

Days

Cell

Num

bers

FIGURE3: A) (FIRST TEST) GROWTH CURVE ANALYSIS OF STERILE WATER TZ EXTRACTS ON A-549 CELLS, STARTING WITH 50,000 CELLS. B) (SECOND TEST) GROWTH CURVE ANALYSIS OF STERILE WATER TZ EXTRACTS ON A-549 CELLS, STARTING WITH 75,000 CELLS.

0 1 2 3 4 5 60

200000400000600000800000

10000001200000140000016000001800000

Control Cells

Treatment Cells

Days

Cell

Num

bers

When testing the Tz extract on another cancer cell line, the SCC-13y’s, results were similar to

what was found with the A-549’s. As seen below in Figure 4, when the SCC-13y’s were treated

with the DMSO Tz extract, the cells experienced the same inhibitory effect. By Day 5, treated

cells had dropped below the expected numbers, with nearly a 200,000 cell difference. This

suggests that the extract caused the cells to undergo some form of cell death, like apoptosis.

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0 1 2 3 4 5 60

50,000100,000150,000200,000250,000300,000350,000400,000450,000500,000

Control Cells

Treatment Cells

Days

Num

ber o

f Cel

ls

FIGURE 4: GROWTH CURVE ANALYSIS OF DMSO TZ EXTRACTS ON SCC-13Y CELLS, STARTING WITH 75,000 CELLS. THE SAME PROTOCOL WAS FOLLOWED AS IN FIGURES 2 AND 3.

Previous studies (Brauner, A. 2012) have been performed on a non-cancerous HFF1 cell line

but with the more crude and un-quantified Tz extract. Figure 5a shows the growth curve

analysis of HFF1 cells that were treated with either water or the Tz extract. Figure 5b shows the

HFF1 cell counts for both the treated and negative control cells on Day 0 and Day 2. These

experiments, though preliminary, have shown there was a significant similarity in how the

normal (non-cancer) cells reacted to the Tz extract.

When treated with the Tz treatment, the HFF1 cells experienced a similar inhibitory effect,

with little to no growth occurring when compared to the negative control cells, which

proliferated normally.

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FIGURE 5: A) GROWTH CURVE ANALYSIS OF A 2.5% TZ AQUEOUS EXTRACT. 62,500 HFF1 CELLS WERE SEEDED, TREATED WITH EXTRACT, AND COUNTED 24 AND 48 HOURS AFTER SEEDING. B) COMPARATIVE ANALYSIS OF 2.5% TZ EXTRACTS ON HFF1 CELLS AS COMPARED TO THE NEGATIVE CONTROL OF STERILE WATER.

Discussion

One of the most promising and significant aspects of this research was the improvement

and finalization of the extract protocols in an effort to create a pure, sterile, and

quantifiable extract. Though both the aqueous and the methanol extracts gave promising

results, we decided that the majority of future testing will be done with a methanol extract

in an effort to preserve the water soluble molecules.

It was found that Tradescantia zebrina extract does have an inhibitory effect on the

cellular growth of both A-549 and SCC13y cancer cells and normal HFF1 cells. However,

extended research with the HFF1 cells is required because the only data collected was

preliminary and involved the use of the crude, less sterile extract. To obtain more relatable

and statistically relevant data, the more purified and quantifiable extract will be used in

future experiments, which will allow for the comparison of the new results to the previous

results gathered. Though preliminary tests held promise, they were never statistically

significant due to the lack of multi-plate testing. However, after implementing a new system of

three plates for each count, it was found that results were still quite similar and also much

more statistically significant.

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There is a great deal more that can be done to explore the value and potential this plant

has to offer, especially when related to anti-cancer activity. One of the major research

methods we want to pursue is flow cytometer readings that include using fluorescence-

activated cancer cells. This would allow us to determine what is happening to the cell cycle

of treated cells and ultimately would clarify any cytostatic or cytotoxic activity occurring.

Another important method that would give a clear view into how cells are being affected

by the Tz treatment would be to perform a clonogenic survival study. This survival assay

determines the ability of a cell to proliferate indefinitely while retaining its reproductive

ability to form a larger colony (SOURCE). In relation to tests done with the Tz extract, a

clonogenic survival assay should be performed in order to determine the survival rate and

reproductive integrity of both the A-549 and SCC13y cells after the Tz treatment is applied

for a few days and then removed.

Finally, it is important to mention previous work done with a crude extract of a close

relative of Tz, known as Tradescantia fluminensis. Work done on SCC-13y cells with this Tf

extract found that similar inhibitory effects were observed (Brauner, A. 2012) when

compared to data collected from Tz tests. This correlation between the two different but

somewhat similar extracts suggests that similar compounds are found in both species of

plants and that these specific compounds could be responsible for the effects on both the

cancer cells and the normal cells. For this reason, more in depth research of Tradescantia

fluminensis should be done to determine if it has anti-cancer properties like Tradescantia

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zebrina and if it is more effective or less effective. Effects of Tf extracts on SCC-13y cells are

shown below in Figure 8.

Figure 8: A) Growth curve analysis of T. fluminensis aqueous extract. 62,500 HFF1 cells were seeded, treated with extract, and counted 24 and 48 hours after seeding. B) Comparative analysis of T. fluminensis extracts on HFF1 cells as compared to the negative control of sterile water.

References

Adhami, Vaqar Mustafa. "Oral infusion of pomegranate fruit extract inhibits prostate carcinogenesis in the TRAMP model." NCBI. US National Library of Medicine National Institute of Health. , 22 Dec. 2012. Web. 4 Oct. 2013. www.ncbi.nlm.nih.gov/pmc/articles/PMC3291862.

"Cancer Facts and Figures 2011." cancer.org. N.p., 12 Dec. 2011. Web. 1 Oct. 2013. <www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-029771.pdf>.

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Pandi, M. "Anticancer activity of fungal taxol deri... [Biomed Pharmacother. 2010] - PubMed - NCBI." National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013. http://www.ncbi.nlm.nih.gov/pubmed/19762199.

Talib , Wamidh H. . "Antiproliferative Activity of Plant Extracts Used Against." Sci Pharm. N.p., n.d. Web. 2 Oct. 2013. http://www.scipharm.at/download.asp?id=606.

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