What is acetaminophen? Acetaminophen (APAP) is the active pharmaceutical ingredient of Tylenol® and other pharmaceutical generics, used as an analgesic. Previous experiments and data has suggested this molecule can potentially induce negative off-target effects in healthy, biological cells and tissues of the human body [1,2,3]. The specific effects discovered, of this small molecule included decreasing cell proliferative function, alter morphology, and omit intercellular protein interactions of normal cells [1,2,3]. If studies can biologically isolate the APAP’s function of causing these biological negative feedbacks, then experimental research on cancer cells should be eminent. It was originally hypothesized that the additive effects of Tylenol®, Advil®, and Aleve®, causes off-target effects on mouse lymphocytic leukemia cells (L1210) and over time, kill off the entire population. It was narrowed down to APAP, having the most extreme and quickest change in this cell’s proliferation and adhesion functions in a given time interval. Immortalized Human T Lymphocytes (Jurkat) were decided on because it needs to be seen if there is a biosimilar effect on a human cancer cell line. Therefore, it was hypothesized that APAP will suppress the Jurkat cell’s proliferative function, alter membrane shape, change the intercellular behavior, and induce apoptosis, due to the highly suggestive evidence that APAP signals to off-target proteins in biological cells. Knowing this information can potentially have researchers and biotech companies alike, further work with APAP and adjust it accordingly, as a potential oncotoxic molecule for cancer therapy.

1. Introduction
What is acetaminophen? Acetaminophen (APAP) is the active
pharmaceutical ingredient of Tylenol®
and other pharmaceutical generics,
used as an analgesic. Previous experiments and data has suggested this
molecule can potentially induce negative off-target effects in healthy,
biological cells and tissues of the human body [1,2,3]
. The specific effects
discovered, of this small molecule included decreasing cell proliferative
function, alter morphology, and omit intercellular protein interactions of
normal cells [1,2,3]
. If studies can biologically isolate the APAP’s function
of causing these biological negative feedbacks, then experimental
research on cancer cells should be eminent. It was originally
hypothesized that the additive effects of Tylenol®,
Advil®,
and Aleve®,
causes off-target effects on mouse lymphocytic leukemia cells (L1210)
and over time, kill off the entire population. It was narrowed down to
APAP, having the most extreme and quickest change in this cell’s
proliferation and adhesion functions in a given time interval.
Immortalized Human T Lymphocytes (Jurkat) were decided on because
it needs to be seen if there is a biosimilar effect on a human cancer cell
line. Therefore, it was hypothesized that APAP will suppress the Jurkat
cell’s proliferative function, alter membrane shape, change the
intercellular behavior, and induce apoptosis, due to the highly suggestive
evidence that APAP signals to off-target proteins in biological cells.
Knowing this information can potentially have researchers and biotech
companies alike, further work with APAP and adjust it accordingly, as a
potential oncotoxic molecule for cancer therapy.
Materials and Methods
Jurkat Cell Culture Stock: A Jurkat cell line was maintained and cultured in
a T75 cell culture-treated flask, using RPMI, FBS (fetal bovine serum), and
penicillin/streptomycin (P/S) antibiotics.
Jurkat Control: Jurkat control was nontreated. The control was treated with
standard Jurkat media of RPMI, FBS, and P/S antibiotic. No APAP.
Jurkat Treated: Jurkat treated contained 25% APAP (W/V), which was
directly mixed in with standard Jurkat media. The purified APAP was
manufactured by Sigma-Aldrich and was suggested to be prepared at 1400
mg/ml solvent (Jurkat media) which created a 1.4% strength.
Automated Cell Counter and Producing a Growth Curve: The
comparative growth curve was produced using the Countess™ II FL by Life
Technologies. Preparation was performed in a 6-Well plate and done in
triplicate for each time interval measured. The triplicate data was averaged.
The average viability count of the Jurkat control and treated, were recorded
and plotted at t = 0, 48, 72, 168, 216, and 240 hours.
Fluorescent Microscopy Imaging: Calcein and ethidium bromide was
prepared in sterile phosphate buffer saline (PBS). The prepared solution was
then aliquoted into the treated and nontreated Jurkat cells, seeded at 50,000
cells/ml. The cells were then incubated in the dark for 25 minutes before
observing it under the fluorescent microscope. Calcein labels the live Jukat
cells and ethidum labels the dead Jukrat cells.
Flow Cytometry Analysis: β-1 integrin, α-3 integrin, and α-5 integrin
antibodies were used on 1 ml Jurkat cell suspensions of the control and
treatment groups. This method was used to measure the amount of protein
expression involved in Jurkat cell-cell adhesion, corresponding to β1, α3, and
α5 integrin proteins in the Jurkat cell membrane. A guava easyCyte ™ flow
cytometer by Millipore was used to produce the dot plot data. 0 hour and 48
hour time intervals of the control and treated Jurkat cells were measured.
Results Conclusions
The hypothesis was confirmed that APAP can potentially suppress Jurkat
cell proliferative function, alter its membrane shape, change its
intercellular adhesion behavior, and induce a faster rate of apoptosis.
Figure 1 Conclusion: The 25% treated had shown a linearization effect,
in which the cells did not have an exponential growth phase within the 9
days of treatment and observation. This suggested that it had decreased
their ability to proliferate through its extracellular and intracellular
signaling mechanisms.
Figure 2 Conclusion: The 25% treated had shown little to no cell-cell
aggregation, and displayed the highest red fluorescence for all times
intervals collected. Qualitatively, this suggested that it had altercated the
cell’s membrane morphology and peripheral proteins involved in cell-cell
adhesion and survivability signaling.
Figure 3 Conclusion: There were 3 variations, in the results collected for
the histogram plot analysis. β-1 integrin, green fluorescence was
instantaneously effected by the APAP at 0 hour and 48 hour analysis. The
α-3 integrin, yellow fluorescence was not effected for 0 hour and 48 hour
analysis and displayed no significant difference. α-5 integrin, yellow
fluorescence did decrease, but the effect of APAP was prolonged. There
was an increase of yellow fluorescence at 0 hour analysis. But after 48
hours, α-5 integrin, started to decrease in yellow fluorescence. All 25%
APAP treated displayed a decrease in cell count on the y-axes.
Figure 4 Conclusion: There were 3 variations, in the results collected for
the dot plot analysis. The cell population in the 25% APAP treated,
tagged for β-1 integrin, displayed an increase in double negative integrin
expression. The cell population in the 25% APAP treated, tagged for α-3
integrin, displayed no significant population change in integrin
expression. The cell population in the 25% APAP treated, tagged for α-5
integrin, displayed a delayed increase of double negative integrin
expression at 48 hour analysis, but not at 0 hour analysis.
Ultimately, this data suggests that APAP can either interfere with either
the β-1 integrin and α-5 integrin proteins, or indirectly through the signal
pathway. APAP altercated the adhesion and survivability of Jurkat cells,
when inducing in its main mechanism of action.
Lance Meitz and Melissa McCoy, MS Biotechnology and Bioinformatics Program Spring 2018
California State University Channel Islands - One University Drive Camarillo CA 93012
References
1. Jack A. Hinson,corresponding author Dean W. Roberts, and Laura P.
James. Mechanisms of Acetaminophen-Induced Liver Necrosis. Handb Exp
Pharmacol. 2010; (196): 369–405.
2. Hartmut Jaeschke Mary Lynn Bajt. Intracellular Signaling Mechanisms of
Acetaminophen-Induced Liver Cell Death. Toxicological Sciences, Volume
89, Issue 1, 1 January 2006.
3. Boulares AH, Zoltoski AJ, Stoica BA, Cuvillier O, Smulson ME.
Acetaminophen induces a caspase-dependent and Bcl-XL sensitive
apoptosis in human hepatoma cells and lymphocytes. Pharmacol Toxicol.
2002 Jan;90(1):38-50.
Future Work
With these assumptions and conclusions, it would need to be further confirmed with
biochemical structure assays, such as x-ray crystallography or NMR Spectroscopy, to
prove that APAP can potentially interact with or signal to off-target proteins.
Attenuation of APAP off-target effects on healthy cells would be the priority, if this
small molecule is ever to be researched on, as an oncotoxic therapeutic. Possible ways
to make APAP target-specific to certain cancers, can include adenovirus drug carriers
or polymer-drug conjugates.
Figure 1: Jurkat Cell Growth Curve (Control vs. 25% Acetaminophen Treated) The graph displays a
significant difference of the 25% APAP treated (W/V) at t = 0, 48, 72, 168, 216, and 240 hours, when
compared to the Jurkat control. No exponential growth phase is observed for the Jurkat cells treated with
25% APAP (W/V). The control displays a sigmoidal-like curve. The 25% APAP treated growth (W/V) is
very linear. Standard Error was included for each individual time point for both the control and the 25%
APAP treated.
Control 25% APAP (W/V) Treated
72 hours
120 hours
144 hours
216 hours
Figure 2: Fluorescent Microscopy of Jurkat Cell Control vs. 25%
Acetaminophen (W/V) Treated at 10x Magnification. The fluorescent
microscope images display a significant morphological and fluorescent change
between the control and 25% APAP (W/V) treated. Fluorescent images were taken
at t = 72, 120, 144, and 216 hours. A difference of Jurkat cell morphological
aggregation between the control and treated are observed. Very little to no cell-cell
aggregation is observed in the treated, when compared to the control. The entire
diameter of the cell-cell aggregated clumps, in the control, are more prominent.
Cell-cell aggregation clumps are observable in the control at all time intervals
observed. In addition, the 25% acetaminophen (W/V) treated Jurkat display more
red fluorescence than the control at all time intervals observed.
Figure 3. Flow Cytometry Data of the 0 hour vs. 48 hours histogram plots of β-1 integrin, α-3
integrin, and α-5 integrin (Control vs. 25% APAP Treated). Blue = Control and Red = 25%
APAP (W/V) Treated. The β-1 integrin displays an instantaneous effect of decreased green
fluorescence for both 0 hour and 48 hour analysis, when treated with 25% APAP (W/V). The α-3
integrin displays no significant difference in yellow fluorescence for both 0 hour and 48 hour
analysis, when treated with 25% APAP (W/V). The α-5 integrin displays a deferred decrease of
yellow fluorescence at 48 hour analysis, when treated with 25% APAP (W/V).
Figure 4. Flow Cytometry Data of the 0 vs. 48 hour dot plots of β-1 integrin, α-3 integrin, and α-
5 integrin (Control vs 25% APAP Treated). The β-1 integrin displays an instantaneous decrease of
green fluorescence in the Jurkat cell population (cell population in single positive quadrant for green
fluorescence skews to double negative quadrant), at both 0 hour and 48 hour analysis. The α-3
integrin displays no significant change in yellow fluorescence among the Jurkat cell population
(population stays within double negative quadrant and single positive quadrant for yellow
fluorescence), at both 0 and 48 hour analysis. The α-5 integrin displays a deferred decrease in yellow
fluorescence in the Jurkat cell population (cell population in single positive quadrant for yellow
fluorescence skews to double negative quadrant) at 48 hours analysis, but not for the 0 hour analysis.
0Hours48Hours
β-1 Integrin α-3 Integrin α-5 Integrin
0 Hours 48 Hours
β-1Integrin
β-1Integrin
α-3Integrin
α-3Integrin
α-5Integrin
α-5Integrin