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The Effects of Wortmannin on DU-145 Cells by
Obstruction of the PI3K-AKT and MAPK Pathways
Madison Lewis, Matthew Rather, Austin Roberson, Kyle Taylor
BMED 3610 - Section C - Fall 2016
Prostate cancer is the second most common malignancy in men
worldwide, with approximately 14% of men becoming diagnosed over
their lifetime. Prostate cancer is also the second leading cause of cancer
death in American men, killing every 1 in 39 men diagnosed[2]
. Even as a
significant source of malignancy, few therapeutics have shown promise
in significantly reducing metastasis. Since its initial discovery as a
proto-oncogene, the serine/threonine kinase Akt has become a major
focus of attention because of its critical role in regulating diverse cellular
functions including metabolism, growth, proliferation, survival,
transcription, and protein synthesis[3]
. Dysregulation of the
phosphoinositide 3-kinase (PI3K/Akt) pathway is implicated in many
human diseases, such as cancer, where the mutations cause an increase
in the intrinsic activity of this pathway. MAPK has also been linked to
tumor growth and apoptosis in cancerous cells[4]
. Introducing
wortmannin, a steroid metabolite of the fungi Penicillium funiculosum,
has shown promise in being able to decrease activity of these pathways
in breast or colon cancers, but is relatively untested against prostate cell
lines. Knowing the prior success of wortmannin in decreasing the
activity of these pathways, it is hypothesized that the addition of
wortmannin to the DU-145 prostate cancer cell line will lead to a
decrease in expression of the PI3K/Akt pathway and possible
interference of the MAPK pathway. Furthermore, the decreased
expression of these paths will lead to an increase in cell death by
apoptosis. To test this, both CCK-8 viability assays and Muse flow
cytometry will be used to determine the morphological characteristics as
well as the overall expression of this pathway before and after treatment.
Hypothesis
Materials and Methods
Citations
Abstract
Introduction
Concentration (nM) 0 (control) 50 100* 250 400
Avg. Value 0.7621 0.6753 0.4186 0.2163 0.2037
Std. Dev 0.1377 0.2339 0.1418 0.0225 0.0079
% Control 100 88.6 54.93* 28.38 26.74
Dual PI3K/MAPK Flow Cytometry
Conclusion
Discussion
CCK-8 Cell Viability Assay Process
CCK-8 Analysis Results
Future Work
Dysregulated signal transduction from receptor tyrosine
kinases to phosphatidylinositol 3-kinase (PI3K) and AKT (protein
kinase B) occurs due to the inactivation of tumor suppressors[1]
. It
has also been discovered that in high concentrations wortmannin
also has shown inhibitory behavior of other PI3K-related enzymes
such as mTOR and MAPK. This disrupted signal transduction is
common among cancer cells. Here, it is demonstrated that the
introduction of the steroid metabolite, wortmannin, affects cancer
growth, specifically in these pathways. The DU-145 prostate cancer
cell line was chosen as a representative cell line and was explored
in many aspects once introduced to wortmannin. Cell viability,
morphological characteristics, as well as specific pathway
activation were all considered when determining the effects of
wortmannin. Results confirmed a decrease in cell viability as well
as PI3K pathway activation among cells treated with wortmannin.
These results suggest wortmannin as an effective therapeutic
agent against the dysregulation of the cell cycle, and directing
apoptotic cell death.
Analysis of the absorbance data collected from the plate reader on the
treated DU-145 cells at various concentrations showed a dose-
dependent relationship, with the highest concentration of wortmannin
showing the least amount of viable cells.
● DU-145 prostate cancer cells
● Eagle’s Minimum Essential Media
● Wortmannin
○ 10 mg acquired from Sigma-Aldrich in powder form
○ Dissolved in DMSO for 1000X dilution and stored at -20° C
● CCK-8 Assay Kit for cytotoxicity assay, plate reader
● PI3K/MAPK Dual Pathway Activation Kit
● Muse Cell Analyzer for flow cytometry
Figure 1: Absorbance
Values of Cells
Treated with
Wortmannin
Results of Dual PI3K/MAPK Flow Cytometry
1. Hua Zhong, Kelly Chiles, et al. Modulation of Hypoxia-inducible Factor 1α Expression
by the Epidermal Growth Factor/Phosphatidylinositol 3-Kinase/PTEN/AKT/FRAP
Pathway in Human Prostate Cancer Cells: Implications for Tumor Angiogenesis and
Therapeutics. Cancer Res March 15 2000 (60) (6) 1541-1545;
2. American Cancer Society. Key statistics for prostate cancer. March, 2016.
3. Cell Signaling Technology. PI3K/ Akt Signaling Pathway Description. 2014.
4. Wada T, Penninger JM. Mitogen-activated protein kinases in apoptosis
regulation. Oncogene. 2004 Apr 12;23(16):2838-49. Review. PubMed PMID:
15077147.
5. Edlind MP, Hsieh AC. PI3K-AKT-mTOR signaling in prostate cancer progression and
androgen deprivation therapy resistance. Asian Journal of Andrology.
2014;16(3):378-386. doi:10.4103/1008-682X.122876.
6. Sigma-Aldrich. Wortmannin from Penicillium funiculosum datasheet.
7. Aksamitiene, E., Kiyatkin, A., & Kholodenko, B. N. (2012). Cross-talk between
mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochemical
Society Transactions,40(1), 139-146.
Table 1: Absorbance Values from CCK-8 Assay Values of absorbance readings of the various
wortmannin treatment concentrations. The IC50
was found to be approximately 100 nM and this
concentration was used for later analysis.
Cells plated in 96-well plate from flask, 5000
cells/well in 200 μL EMEM/well, 15 wells
Cells incubated for 24 hours
Cells treated in triplicate with the following
treatments: Control (0 nM), 50 nM, 100 nM, 250
nM, 400 nM
Cells incubated for 48 hours
CCK-8 solution added from kit, incubated 1 hour
Absorbance measured in plate reader at 450
and 650 nm, data analyzed
Cells plated in 6-well plate from flask, 75000
cells/well in 3 mL EMEM/well, 6 wells
Cells incubated for 24 hours
Cells treated in triplicate with either
control (0 nM) or 100 nM, which was
found as IC50
Cells incubated for 48 hours
Cells trypsinized and pooled by treatment group
Flow cytometry performed in Muse Cell
Analyzer as instructed by user guide
Wortmannin will have a dose-dependent effect on DU-145 cells,
which will be seen in the cytotoxicity assay. In addition, wortmannin
will cause inactivation of the PI3K signaling pathway and cause cell
death by apoptosis, with evidence of the MAPK pathway also being
obstructed.
Wortmannin is a potent PI3K inhibitor that has shown promise in its
derivatives of treating malignant cancers. Prostate cancer growth is linked
heavily to the PI3K pathway, with evidence of cross-talk to the MAPK
pathway. Because wortmannin acts on this pathway it is logical to attempt to
develop new treatments based on its properties.
Our results show that high concentrations of wortmannin do cause cell
death. The phase contrast images provide evidence into the method in which
the cells died. Lack of cell debris within the media suggests the cells
underwent apoptosis instead of lysing from wortmannin exposure, which
may support claims that the MAPK pathway is linked to apoptosis when also
compared to the flow cytometry data. There was significant decrease in the
PI3K only activation when exposed to wortmannin, but dual PI3K/MAPK
activation was increased without much MAPK only activation, possibly also
meaning wortmannin causes an increase in the cross-talk among the
pathways.
Future studies should be conducted on DU-145 cells and the effects
of wortmannin to gain better understanding of why the MAPK and dual
pathways showed increased activation. Testing should be done with a
known MAPK inhibitor to understand if PI3K activity was inhibited or only
an increase in cross-talk occurs. It should also be examined why there
was a small, but present increase in the single activation of the MAPK
pathway. A western blot could also be used to examine MMP-9, NF-κB
p65, and β-actin (as a loading control) to quantify the proliferation effects
of wortmannin. This knowledge of pathway activation could be useful not
only for prostate cancer, but many metastatic cancers.
The CCK-8 cell viability assay results were as expected. They
showed that wortmannin has a dose-dependent cytotoxic effect on
DU-145 cells, with an IC50
of approximately 100 nM. This
concentration was used as the treatment group for flow cytometry.
The Muse analysis results were somewhat interesting. The
Muse Dual PI3K/MAPK Activation Kit showed that wortmannin
caused a decrease in single activation of the PI3K pathway, an
increase in the dual PI3K/MAPK pathway activation, and a small
increase in the MAPK pathway activation. As seen in Figure 5,
there is little cell debris seen. This suggests that wortmannin may
have an impact on the cross-talk present within DU-145 cells and
may have caused an increase in apoptosis due to the increase in
both MAPK single activation and MAPK within the dual activation
group as well.
Figure 3: Phase contrast image of DU-145
Control Cells This image shows untreated
DU-145 cells after incubating for 48 hours in a
96-well plate. The cells have grown tremendously
and cover the plate, making individual cells nearly
indistinguishable.
Figure 2: Results of Dual PI3K/MAPK Pathway
Activation Kit with Control (0 nM) wortmannin
Concentration First, cells were sized in order to
eliminate debris from the analysis. On the second
chart, it can be seen that most activation was
present in the PI3K pathway alone. Dual activation
was also present. MAPK signal pathway activation
was not activated.
Figure 4: Results of Dual PI3K/MAPK Pathway
Activation Kit with IC50
(100 nM) wortmannin
Concentration Cells were sized in order to
eliminate debris from the analysis. It can be seen
in the second chart that most activation was dual
PI3K/MAPK, increasing almost 21%. PI3K
activation did decrease by 14%. MAPK signal
pathway activation was expressed in a very small
amount.
Figure 5: Phase contrast image of DU-145 IC50
Cells This image shows DU-145 cells treated with
100 nM wortmannin after incubating for 48 hours
in a 96-well plate. The cells have grown, but are
not quite as plentiful as the control group. Gaps
are present in the covering of the cell surface, and
individual cells can be seen. There is little debris
present, which may be evidence of apoptosis.