The resulting research poster from my internship with Helios Scholars at TGen.

1. Translational Genomics Research Institute | www.tgen.org
The Effects of Natural Compounds on H4 Neuroglioma Cells
Tingting Thompson1, Jerry Antone2, Leslie Gunatilaka3, Winnie S. Liang2
1Helios Scholars at TGen, 2Translational Genomics Research Institute, 3University of Arizona
METHODS
Figure 1.1 75 cm2 cell culture flask
used in project
1. Astashkina, A., Mann, B., & Grainger, D. W. (2012). A critical evaluation of in vitro
cell culture models for high-throughput drug screening and toxicity. Pharmacology &
Therapeutics,134(1), 82-106. doi:10.1016/j.pharmthera.2012.01.001
2. Mangal, M., Sagar, P., Singh, H., Raghava, G. P. S., & Agarwal, S. M. (2013).
NPACT: Naturally Occurring Plant-based Anti-cancer Compound-Activity-Target
database. Nucleic Acids Research, 41(Database issue), D1124–D1129.
3. Newman, D. J., & Cragg, G. M. (2012). Natural Products As Sources of New Drugs
over the 30 Years from 1981 to 2010. Journal of Natural Products,75(3), 311-335.
doi:10.1021/np200906s
REFERENCES
In vitro cell culture serves to predict the effects of test compounds as drug sources
for in vivo application, permitting an intermediate step before clinical trials. Popularity of
in vitro drug screening stems from the ability for scientists to observe the effects of
compounds on the cells of interest in an efficient and lower-risk manner1.
The mass of data relevant to cytotoxicity studies of natural compounds has been
compiled into 1574 entries in a resource known as the Naturally Occurring Plant-based
Anti-cancer Compound-Activity-Target database2. Natural product drug discovery
exhibits great promise, especially since, from the 1940’s to 2010, 48.6% of the cancer
drugs were either derived from natural products or were natural products themselves3.
Rather than specifically target a compound based on its chemical properties, we
blindly tested eighty-eight plant-derived natural compounds from a collaborative center
run by Dr. Gunatilaka at the University of Arizona.
INTRODUCTION
Example: Oncovin®
 Vincristine Chemical
 Extracted from the Madagascar
Periwinkle, Catharanthus Roseus
 Chemotherapy that treats leukemia,
lymphomas, advanced testicular
cancer, breast and lung cancers,
and Kaposi's sarcoma
Cell Treatment
❖ Dilutions (in DMSO and media):
10uM→1mM→100uM→5uM
❖ Five plates of cells
WORKFLOW
Cell Culture
❖ Grown in 20mL media and growth
serum solution, without antibiotics
❖ Adherent cells uplifted using Trypsin
❖ Plated during passage 6-10
Figure 1.3 Cell plate organization with
crystal violet solution
❖ PBS loaded in exterior wells to limit
effects of evaporation
❖ Compounds added in triplicate
Crystal Violet Assay
 Evaluates cell viability and compound
cytotoxicity
 Crystal violet stains DNA of viable cells
 Washes help to dislodge dead cells and
measure the most accurate reading
Analysis
 Higher levels of absorbance = more
staining = greater level of cell viability =
lower levels of compound cytotoxicity
 Compare cytotoxicity levels to that of
digitonin, the positive control and a
known cytotoxic compound that helps us
establish a significance level
The identification and development of novel treatment approaches for cancer
continues to be a pressing area of research. While newer strategies such as
immunotherapy has demonstrated efficacy in specific cancers, continued interrogation
of new compounds is needed to identify potential new avenues to combat disease.
Notably, over a quarter of drugs used for treating cancer are extracted from plants, and
approximately a quarter of cancer drugs are modified extracts from plants. With the
goal of identifying novel anti-carcinogenic compounds, we tested the effects of eighty-
eight natural plant-derived compounds on H4 Neuroglioma cells. We first determined
the most optimal approach for accurately measuring the cytotoxic effects of the
compounds, and using a crystal violet assay, we tested the post-treatment cell viability
and corresponding drug cytotoxicity of these compounds. This assay stains DNA in
viable cells and thereby allows us to evaluate which compounds are more cytotoxic.
Characterizing and understanding the effects of these natural compounds will allow us
to determine if any may represent potential leads to enable drug discovery efforts
aimed at treating brain tumors and improving outcomes for patients.
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Figure 1.2 Image of 96-well plate with diluted test compounds
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Figure 1.3 Image of plated cells with assigned triplicate
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ABSTRACT
1. Seed Cells
 Cells loaded in 200uL of media per well
 Cells seed for at least 24 hours
 Grown until each well is about 60-80% confluent
with cells
2. Stimulate Cells
 Controls (100uL): staurosporine (10uM), digitonin
(30ug/mL), DMSO (0.5%), no treatment (only
media), and no cells (only media)
 88 compounds at 5uM of 100uL solution
3. Stain and Wash Cells
 0.5% crystal violet staining solution
 Enough solution to cover well, ~35uL
 Wash with water before and after staining
4. Add Methanol and Measure OD
 50uL methanol added for 20 minutes
 OD measured at 595nm
5. Compare Optical Densities
 Read absorbance/ OD
 Normalized OD using no-cell wells
Normalized OD= ODexperimental- ODno cell
 Evaluated cytotoxicity using no-treatment
(ODno treatment-ODexperimental)
ODno treatment
Figure 1.5 BioTek Cytation 3 Cell Imaging
Reader used to measure optical density (OD)
Round 1
Although a large number of our compounds showed greater cytotoxic effects than our
positive control, digitonin, we were concerned by the lack of variation in OD between
the no-cell and no-treatment wells. We then suspected the no-treatment control and
other wells were inaccurately reflecting viability, perhaps due to a loss of cells during
staining.
Troubleshooting
After visualizing wells under the microscope, we confirmed the loss of cells during the
process. We hypothesized that we were losing cells during the washing and/ or
aspirating steps in the staining process.
What did we test?
Varied the aspiration technique during wash before and after staining:
1. Low pressure glass aspirator without tip + wrist flick
2. High pressure glass aspirator without tip + P200 multichannel removal
3. High pressure glass aspirator with tip + wrist flick
4. P200 multichannel removal + multichannel
5. Submersion in water + wrist flick
Decision: gently load water and wrist flick
Rounds 2-4 (Troubleshooting)
The cytotoxicity levels of greater than 100% corresponds to a negative
normalized OD, which can only occur if the no-cell wells had a greater absorbance
than the experimental wells.
In theory, the no-cell wells should have the lowest OD; it is troubling to continue
to have the OD of no-cell wells be similar to OD of no-treatment. Both concerns have
led us to believe there is more troubleshooting to be done.
What could have gone wrong?
If a well lost viable cells, not due to the compound, the absorbance would be
lower. Varying levels of absorbance between wells within the same triplicate might
have stemmed from an ”edge effect”, when more crystal violet residual remains in the
center wells during washing. The no-treatment wells were plated on the edge by the
no-cells and might have lost cells, creating the illusion of similar OD’s.
Future Directions
Due to the initial inability to keep viable cells in the 96-well plates, we were unable to
confidently conduct the crystal violet assay. Continued testing will be required to
accurately characterize the cytotoxicity of the compounds.
Next steps:
1. Continue troubleshooting, possibly try using six-well plates that do not hold onto
liquid as tightly.
2. Run crystal violet assays using a protocol that has been adjusted to limit loss of
viable cells.
3. Evaluate apoptosis along with cytotoxicity and viability using Apotox- Glo assay.
• Triplex assay with three reagents to track cell viability, drug cytotoxicity, and
apoptosis activity.
• Staurosporine acts as positive control due to its association with apoptosis.
DISCUSSION
RESULTS
% Cytotoxicity= • 100
Thank you to the Helios Education Foundation and the Helios Scholars at TGen for the
opportunity to have such a wonderful experience. I also wanted to thank Dr. Winnie
Liang and the Liang Lab (Jon Adkins, Phillip Geiger, Lori Cuyugan, Erica Tassone) for
hosting me this summer, especially my day-to-day mentor, Jerry Antone. Additionally, for
all the support, I thank my friends, family, and other Helios interns.
ACKNOWLEDGEMENTS
Figure 3.1 Image of the top of stained well, aspirated using no
tip, high pressure glass pipette
Figure 3.2 Image of the bottom of stained well, aspirated
using no tip, high pressure glass pipette
1 2 3 4 5 6 7 8 9 10 11 12
A 0.068 0.074 0.048 0.068 0.048 0.049 0.049 0.058 0.049 0.049 0.048 0.048
B 0.062 0.086 0.14 0.195 0.238 0.298 0.213 0.214 0.149 0.166 0.067 0.048
C 0.051 0.186 0.271 0.28 0.261 0.265 0.227 0.222 0.17 0.181 0.115 0.049
D 0.049 0.162 0.236 0.33 0.342 0.346 0.256 0.233 0.268 0.249 0.164 0.05
E 0.049 0.171 0.23 0.338 0.428 0.345 0.274 0.23 0.24 0.242 0.184 0.048
F 0.052 0.129 0.181 0.202 0.315 0.308 0.221 0.257 0.179 0.173 0.113 0.049
G 0.052 0.059 0.132 0.135 0.179 0.235 0.384 0.19 0.184 0.178 0.059 0.055
H 0.072 0.049 0.049 0.049 0.048 0.07 0.05 0.049 0.049 0.051 0.05 0.048
Figure 3.3 Plate exhibiting the edge effect on the optical densities, resulting from the lack of even crystal violet washing. Darker colors are more stained.
Figure 2.1 Crystal violet assay workflow
0
50
100
150
200
250
300
350
400
Compounds "More Cytotoxic" than DMSO Treatment
-90.00
-70.00
-50.00
-30.00
-10.00
10.00
30.00
50.00
Cpd40
Cpd60
Cpd30
Cpd39
Cpd29
Cpd50
Cpd27
Cpd28
Cpd26
Cpd21
Cpd36
Cpd38
Cpd22
Cpd25
Cpd23
Cpd24
Cpd37
Cpd35
Cpd31
Cpd33
Cpd34
Cpd32
Cpd71
Cpd61
Digitonin
LevelofCytotoxicity
Compounds with Levels Less than Digitonin
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Digitonin
Cpd10
Cpd7
Cpd9
Cpd6
Cpd11
Cpd2
Cpd4
Cpd19
Cpd5
Cpd90
Cpd1
Cpd74
Cpd3
Cpd8
Cpd12
Cpd13
Cpd18
Cpd42
Cpd43
Cpd51
Cpd80
Cpd14
Cpd16
Cpd17
Cpd41
Cpd44
Cpd46
Cpd52
Cpd58
Cpd45
Cpd53
Cpd59
Cpd62
Cpd77
Cpd81
Cpd20
Cpd47
Cpd48
Cpd49
Cpd54
Cpd75
Cpd76
Cpd82
Cpd56
Cpd57
Cpd79
Cpd84
Cpd55
Cpd83
Cpd86
Cpd15
Cpd78
Cpd85
Cpd70
Cpd73
Cpd88
Cpd63
Cpd66
Cpd68
Cpd87
Cpd72
Cpd64
Cpd65
Cpd69
Cpd67
LevelofCytotoxicity
“Significant” Compounds (Greater Cytotoxicity than Digitonin’s)
-110
-60
-10
40
90
140
cpd10
cpd50
cpd30
cpd70
cpd60
cpd20
cpd80
cpd49
cpd48
cpd11
cpd59
cpd40
cpd58
cpd1
cpd47
cpd56
cpd41
cpd46
cpd9
cpd57
cpd79
cpd43
cpd69
cpd55
cpd44
cpd45
cpd8
cpd51
cpd78
cpd68
cpd42
cpd54
cpd39
cpd21
cpd19
cpd29
cpd77
cpd7
cpd67
cpd31
cpd75
cpd28
cpd53
cpd66
cpd65
cpd71
cpd38
cpd61
cpd6
cpd18
cpd12
cpd52
cpd2
DMSO
Compounds "Less Cytotoxic" than DMSO Treatment
Round 1
Round 2-4 (averaged values)
LevelofCytotoxicityLevelofCytotoxicity