This document summarizes an experiment that tested the effects of amygdalin (B17) on cervical cancer (HeLa) cells at various concentrations and incubation times. HeLa cells were cultured with B17 at concentrations of 1.25, 2.5, 5, 10, and 20 mg/mL for 24, 48, and 120 hours. An MTT assay was then used to analyze cell viability. The results showed that 10 and 20 mg/mL B17 concentrations caused significant HeLa cell apoptosis after 120 hours of incubation. Lower concentrations had little effect even after longer incubations. Initial issues with low cell density required troubleshooting to obtain clear results from the MTT assay.

1. HELA CELL GROWTH
AND PROLIFERATION
WITH B17-AMYGDALIN
By Jason Morris of Minneapolis Community and
Technical College, 1501 Hennepin Avenue,
Minneapolis, MN, 55403
ABSTRACT
Amygdalin (B17) is known to have some anticancer
properties and even to target and destroy cervical
cancer cells (1). The aim of this experiment was to
confirm the extent of the above claim. Not only that
B17 was cytotoxic to cells or not was of primary
interest, but at what concentrations and for how long
the cells were under such conditions as well.
Mammalian cell-line HeLa cells were monolayer
cultured in EMEM in T-25 flasks to above 90%
confluency before removing media and undergoing
B17 treatment with media for 24 hours of varying
concentrations. After 24 hours, the B17 and media
were removed and cells were analyzed via MTT assay
method using D-PBS. The above steps were carried
out at incubation times of 48, 72, and 120 hours as
well. After troubleshooting various obstacles, the
results showed that optimal concentrations of B17 for
HeLa cell apoptosis were 10mg/mL and 20mg/mL for
120 hours incubation time. Use high cell density for
B17 on HeLa cells or results will need troubleshooting.
Conducted April 7th-April 27th, 2015
In partial fulfillment of Cell Culture Techniques
Course Project under the supervision of Dr. Rekha
Ganaganur, spring semester, 2015

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Introduction:
MTT assay is a method often used by scientists interested in culturing cell lines. It is a means of
quickly determining cell viability, proliferation, and activation of cells without having to harvest and
count them individually.2
2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is
reduced by mitochondrial dehydrogenase enzyme under normal cell conditions. The reduced product is
purple/blue and called formazan. Under normal conditions, when not reduced,it is a yellow, soluble
substrate. It can be used to differentiate between proliferation and cell activation and can be used on
monolayer and suspension culture. In the case of this experiment, monolayer HeLa cells were used with
MTT assay.
Amygdalin, or B17, is a naturally occurring substance derived from seeds of apricots, almonds,
peaches,apples, and several other members of the prunasin family.(1,3)
It is a cyanide former and has been
known to cause apoptosis (programmed cell death) of cervical cancer cell-line HeLa cells.1
Amygdalin is
of growing interest to cancer biologists and researchers of multiple other disciplines. The mode of action
of amygdalin on cervical cancer cells are unknown still.3
It is possible to gain further insight into this
looming question by subjecting HeLa cells with B17 in vitro.
HeLa cell-line was used for this experiment because they proliferate very well for mammalian
cells, especially a human cell line. They are derived from cancer cells of the cervix of one Henrietta
Lacks. This cell line is so resilient, it has been used to develop the polio vaccine, exposed to radiation,
and even shot into space.4
These among other reasons the HeLa cell-line was chosen for this experiment.

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Materials and Methods:
Passage number 7 of HeLa cell-line was harvested for this experiment at approximately 95%
confluency in the T-25 flasks used. Using a 96 well plate, 1.5*104
cells in 150 uL were plated per well.
B17 was prepared by grounding 100mg tablets with mortar and pestle to a fine powder. Starch in the
tablets were removed from the stock solution by centrifuging and removing the supernatant that contained
soluble B17. Working concentrations of B17 were made for 1.25mg/mL, 2.50mg/mL, 5.0mg/mL,
10mg/mL, and 20mg/mL. It was solubilized in sterile EMEM media and filtered with a 0.22um pore size
membrane filter. The 96 well plating format utilized triplicates of all wells. The controls lacked B17
entirely, but included all other ingredients. The remaining 15 wells in each plate had media, cells, aimed
concentration of B17, and eventually MTT to analyze cytotoxicity and other aspects. The plating plan is
as follows:
B17
control
B17
control
B17
control
1.25mg/mL1.25mg/mL1.25mg/
mL
2.50mg/
mL
2.50mg/
mL
2.50mg/m
L
5.0mg/mL5.0mg
/mL 5.0mg/mL
10.0mg/
mL
10.0mg/
mL
10.0mg/
mL 20.0mg/mL20.0mg/mL20.0mg/mL
The above plating plan was for each plate used. Total of three plates used were 24, 48, and 120
hour exposures to B17. The plates were plated and allowed to adhere and the B17 was added. MTT was
added at time of desired exposure after removing the old media. 15mg of MTT was dissolved in 3mL of
PBS. 50uL MTT was added and let incubate for up to two hours. When formazan forms purple color a
microplate could be read at 570nm wavelength.

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Results:
24 hour incubation before addition of DMSO 24 hour incubation after addition of DMSO
48 hour incubation before addition of DMSO 48 hour incubation after addition of DMSO
120 hour incubation before addition of DMSO 120 hour incubation after addition of DMSO

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HeLa cells 24 hour incubation without B17 HeLa cells 24 hour incubation with 20mg/mL B17
Microplate reading after24 hour incubation
Plate Repeat End time
Start
temp.
End
temp. BarCode
1 1
5:40:30
PM 19.9 19.9 N/A
Absorbance @ 570 (0.1s) (A)
0.000
0.392 0.359 0.488 0.413 0.382 0.373 0.228 0.327 0.428 0.468 0.477 0.457
0.297 0.224 0.305 0.275 0.409 0.385 0.399 0.398 0.406 0.265 0.361 0.344
0.036 0.037 0.037 0.037 0.037 0.037 0.036 0.036 0.037 0.037 0.037 0.038
0.036 0.037 0.037 0.037 0.036 0.036 0.036 0.037 0.037 0.038 0.038 0.038
0.036 0.040 0.037 0.036 0.041 0.044 0.043 0.037 0.041 0.041 0.041 0.037
0.035 0.036 0.037 0.037 0.037 0.041 0.037 0.037 0.037 0.038 0.037 0.037
0.036 0.040 0.040 0.037 0.037 0.038 0.041 0.037 0.038 0.037 0.038 0.038
0.036 0.038 0.040 0.037 0.038 0.037 0.038 0.037 0.038 0.039 0.037 0.040
Microplate reading after48 hour incubation
Plate Repeat End time
Start
temp.
End
temp. BarCode
1 1
5:43:45
PM 19.9 19.9 N/A
Absorbance @ 570 (0.1s) (A)
0.000
0.035 0.378 0.045 0.153 1.350 0.489 0.482 0.774 0.049 0.314 0.035 0.133
0.972 0.267 0.359 0.533 0.997 0.357 0.361 0.571 0.688 0.176 0.188 0.350
0.039 0.036 0.037 0.037 0.036 0.036 0.037 0.036 0.037 0.040 0.039 0.043
0.036 0.037 0.037 0.036 0.037 0.038 0.037 0.036 0.036 0.038 0.038 0.039
0.036 0.037 0.039 0.045 0.040 0.046 0.043 0.044 0.041 0.037 0.039 0.038
0.035 0.036 0.037 0.036 0.037 0.037 0.037 0.037 0.037 0.038 0.037 0.037
0.036 0.036 0.036 0.037 0.038 0.037 0.038 0.037 0.037 0.037 0.043 0.040
0.036 0.037 0.045 0.036 0.036 0.043 0.040 0.037 0.036 0.038 0.038 0.048

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Microplate reading after120 hour incubation
Plate Repeat End time
Start
temp.
End
temp. BarCode
1 1
5:45:54
PM 19.9 20 N/A
Absorbance @ 570 (0.1s) (A)
0.000
2.780 2.552 2.708 2.680 2.751 2.038 2.120 2.303 2.456 2.220 2.481 2.386
0.819 1.988 1.455 1.421 0.075 0.096 0.110 0.094 0.082 0.085 0.086 0.084
0.035 0.036 0.039 0.041 0.037 0.036 0.037 0.038 0.037 0.037 0.037 0.036
0.045 0.036 0.038 0.037 0.037 0.036 0.037 0.037 0.037 0.039 0.039 0.036
0.032 0.039 0.037 0.038 0.043 0.041 0.039 0.036 0.038 0.039 0.033 0.034
0.039 0.040 0.041 0.037 0.037 0.035 0.038 0.038 0.039 0.037 0.039 0.036
0.036 0.036 0.038 0.037 0.037 0.038 0.037 0.037 0.037 0.034 0.035 0.036
0.034 0.035 0.036 0.037 0.037 0.038 0.038 0.037 0.038 0.038 0.037 0.036
Averagesof triplicate absorbance readings
Time
incubated
Control
avg
1.25mg/mL
B17 avg
2.50mg/mL
B17 avg
5.0mg/mL
B17 avg
10.0mg/mL
B17 avg
20.0mg/mL
B17 avg
24 hours 0.413 0.327 0.457 0.275 0.398 0.344
48 hours 0.153 0.774 0.133 0.533 0.571 0.350
120
hours
2.680 2.303 2.386 1.421 0.094 0.084
0
0.5
1
1.5
2
2.5
3
0 5 10 15 20 25
Absorbance
B17 concentration (mg/mL
Absorbance vs. B17 Concentration
24 hour exposure 48 hour exposure 120 hour exposure

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Discussions and Conclusions:
Since amygdalin is soluble in media and water,additional solvent was unnecessary. Solvent
controls were therefore not used. Differences between drug control wells and wells containing B17 were
not observed in 24 hour incubation plate. The same results were observed for the 48 hour incubation with
the exception of the 20mg/mL concentration of B17. It appeared that there was slight cell apoptosis in that
range. Similar results observed on 120 hour incubation plate with the exception of 10 and 20mg/mL B17
concentrations where cell apoptosis was abundant. This was indicated by the observation of much lighter
color of wells on 10 and 20 mg/mL versus the deep purple on the controls and other wells, indicating
those cells are proliferating well. The results matched expectations in the sense that as incubation and
concentration increased,the cyanide formed should cause cell increased apoptosis. Overall there were not
unusual results.
There was much troubleshooting needed to gain the proper results, however. For the first two
days of incubation, there was no color whatsoever when MTT was added. So when 120 hours came, Dr.
Rekha, technician Shequaya along with other technicians attempted severalmethods of determination of
why MTT was not producing color change. After changing from D-PBS to PBS,using DMSO to open
cells up, and using confirmatory sf-9 cells it was determined that the MTT itself was not an issue. A
young, bright technician by the name of Cody examined them under a hemacytometer and compound
microscope and found that purple color indeed existed. It was determined by Dr. Rekha after 122 hours of
incubation that the problem lied not in reagent,method, or technician, but that the number of cells plated
(cell density) was far too low, not allowing the naked eye to detect a visible color change. Therefore for
future experiments with cell-line HeLa technicians must use at least 1.5*105
or 1.5*106
cells per well.

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Acknowledgements:
I would like to thank Dr.Rekha Ganaganur for her hard work and dedication to the academic
affairsof her classes and the success of her students. I would also like to thank Shequaya Broadus for her
contributionsto the bettering of this laboratory throughout the semester.
-Jason M Morris

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Sources:
(1) Chen, Yu, Jinshu Ma, Fang Wang, Jie Hu, Ai Cui, Chengguo Wei, Qing Yang, and Fan Li.
"Amygdalin Induces Apoptosis in Human Cervical Cancer Cell Line HeLa
Cells." Immunopharmacology and Immunotoxicology 35.1 (2013): 43-51. Web. 21 Apr. 2015.
(2) Vega-Avila E, Pugsley MK (2011) An overview of colorimetric assay methods used to assess
survival or proliferation of mammalian cells. Proc West PharmacolSoc 54: 10–14.
(3) Chang, Hyun-Kyung, Mal-Soon Shin, Hye-Young Yang, Jin-Woo Lee, Young-Sick Kim,
Myoung-Hwa Lee,Jullia Kim, Khae-Hawn Kim, and Chang-Ju Kim. "Amygdalin Induces
Apoptosis through Regulation of Bax and Bcl-2 Expressions in Human DU145 and LNCaP
Prostate Cancer Cells." Biological & Pharmaceutical Bulletin 29.8 (2006): 1597-602. Web. 21
Apr. 2015.
(4) Ciaccia L. The Immortal Life of Henrietta Lacks. The Yale Journal of Biology and Medicine.
2010;83(3):165.
(5) Li L-Z, Deng H-X, Lou W-Z, et al. Growth inhibitory effect of 4-phenyl butyric acid on human
gastric cancer cells is associated with cell cycle arrest. World Journal of Gastroenterology : WJG.
2012;18(1):79-83. doi:10.3748/wjg.v18.i1.79.
(6) LaPensee EW,Tuttle TR, Fox SR, Ben-Jonathan N. Bisphenol A at Low Nanomolar Doses
Confers Chemoresistance in Estrogen Receptor-α–Positive and –Negative Breast Cancer
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(7) Liu J-J, Zhang Y, Guang W-B,Yang H-Z, Lin D-J,Xiao R-Z. Ponicidin Inhibits Monocytic
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