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Characterization of Apoptotic Pathways Induced in Novel
Photodynamic Cancer Treatment
Kendra Hanslik, Justin Avila, Jennifer Watts, Matthew Gdovin
Department of Biology, The University of Texas at San Antonio, San Antonio, TX. 78249
Abstract
Current cancer chemotherapeutics induce programmed cell death through intrinsic and/or extrinsic
apoptotic pathways. Our lab has created a photodynamic therapy that uses a photo isomerizing
compound (PIC) and UV light to induce cell death. The PIC passively diffuses into cancer cells
and is then activated with UV light, which causes PIC to release H+ ions, lower the intracellular
pH (pHi), and induce apoptotic cell death in vitro. However, it is not clear which apoptotic
pathway the cancer cells follow after receiving our photodynamic therapy. With the use of western
blotting and apoptotic biomarkers, we can characterize the pathway of apoptosis induced by our
treatment. Whole cell lysates will be collected from a breast cancer cell line, MCF-7, exposed to
the following treatment conditions: cells that receive no treatment, which serves as the negative
control, cells treated with ethanol to induce intrinsic apoptosis, cells treated with genistein to
induce the extrinsic pathway, cells treated with luteolin to induce both pathways, and cells treated
with the photodynamic therapy. The cell lysates will then be run on an SDS-PAGE gel and
transferred to a PVDF membrane for western blot analysis using Fas, Procaspase-8, Bax, and
Bcl-2 antibodies. We predict that the cells treated with the photodynamic therapy will show
expression of Bax and little to no expression of Bcl-2, which are two apoptotic proteins expressed
in the intrinsic pathway.These results will provide insight into the apoptotic mechanism that the
photodynamic therapy induces in MCF-7 cells.
Purpose and Hypothesis
The purpose of our experiment is to characterize the apoptotic pathway
induced by our photodynamic treatment in vitro.
We hypothesize that with our photodynamic treatment activating the PIC will
induce intracellular apoptosis via the intrinsic pathway, which would lead to
the expression of pro-apoptotic proteins in this pathway including Bax and
little to no expression of anti-apoptotic proteins such as Bcl-2.
Materials/Methods
Hypothesized Results Discussion
References
Acknowledgements
The results obtained from these experiments will provide insightful
information on which apoptotic pathway our photodynamic treatment
induces in vitro. Previous studies have shown that one method of
characterizing apoptosis is to make an SDS-PAGE gel, transfer the gel to a
membrane, and perform a western blot analysis for specific antibodies (4, 6,
8). To ensure a particular protein is present on the SDS-PAGE gel, an
immunoblotting technique that incorporates western blotting will be used.
Depending on the proteins that are expressed in the blot, we can determine
the primary apoptotic pathway of our photodynamic treatment. If the western
blot shows all apoptotic proteins, it can be proposed that our therapy induces
both pathways. If only Bax and little or no Bcl-2 are expressed, it can be
predicted that only the intrinsic pathway is induced. On the other hand, if
Procaspase-8 and Fas are expressed it can be predicted that the cell is dying
through the extrinsic apoptotic pathway. Thus, it is proposed that the
photodynamic therapy western blot will resemble the western blot from the
EtOH treatment.
While this seems to be a promising method for characterizing an apoptotic
pathway, some limitations regarding SDS-PAGE and western blotting need
to be considered. SDS-PAGE does not definitively identify a specific protein
in that one protein band can be a combination of similarly weighted protein
species. In addition, glycoproteins and acidic residues abnormally migrate in
an SDS-PAGE gel due to the protein’s interactions with the SDS gel
medium, which may lead to a misinterpretation of the protein separation
results (7). While analyzing the western blots, it has been discovered that
phosphorylated or methylated (modified) proteins may not be detected on the
blot when probing with antibodies (1). Lastly, it should be noted that in one
study the Bax protein could be activated indirectly in the extrinsic pathway
(2).
Even after taking these limitations into consideration, this study’s end results
will provide a glimpse into better understanding which apoptotic pathway
our photodynamic treatment induces.
1. Brennan, John. “The Disadvantages of Western Blotting.” eHow. Demand Media Inc., n.d. Web.
5 Oct. 2015.
2. Dewson, Grant and Ruth M. Kluck. “Mechanisms by which Bak and Bax permeabilise
mitochondria during apoptosis.” Journal of Cell Science. 122 (2009): 2801-2808. Web. 6 Oct.
2015.
3. Garfin, David E. Davey, John and Mike Lord, eds. “Gel Electrophoresis of Proteins.” Essential
Cell Structure, A Practical Approach (2003): 197-268. PUBMED. Web. 5 Oct. 2015.
4. Aditi A. Kapasi et al. “Ethanol promotes T cell apoptosis through the mitochondrial pathway.”
Immunology. 108 (2003): 313-320. Web. 20 Sept. 2015.
5. Li, Ruiqi, Navam Hettiarachchy, and Mahendran Mahadevan. “Apoptotic Pathways in Human
Breast Cancer Cell Models (MCF-7 and MDA-MB-231) Induced by Rice Bran Derived
Pentapeptide.” International Journal of Research in Medical and Health Sciences. 4.5 (Sept.
2014): 13-21. Web. 19 Sept. 2015.
6. Hye Sook Seo et al. "Phytoestrogens Induce Apoptosis via Extrinsic Pathway, Inhibiting
Nuclear Factor-κB Signaling in HER2-overexpressing Breast Cancer Cells.” Anticancer
Research. 31 (2011): 3301-3314. Web. 20 Sept. 2015.
7. Roth, Wilfried and John C. Reed. “Figure 1. Pathways for caspase activation and apoptosis.”
Nature Medicine. 8 (2002): 216. Web. 5 Oct. 2015.
8. Shi, Qinwei and George Jackowski. B.D. Hames, ed. “5.6.4 Problems with molecular weight
estimation.” Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed. New York: Oxford
UP, 1998. Web. 5 Oct. 2015.
9. So-hu Park et al. “Luteolin Induces Cell Cycle Arrest and Apoptosis Through Extrinsic and
Intrinsic Signaling Pathways in MCF-7 Breast Cancer Cells.” Journal of Environmental
Pathology, Toxicology, and Oncology. 33.3 (2014): 219-231. Web. 5 Oct. 2015.
Introduction
Development of Treated MCF-7 Cell Lines
Figure 2. A prediction of the results from
the SDS-PAGE assay
Separation of Cell Lysate Proteins
Today, breast cancer is still considered to be one of the leading contributors of
cancer death among women in the United States (8). The therapies available to
treat breast cancers rely heavily on inducing either the intrinsic or extrinsic
pathway of apoptosis. The activity of the extrinsic or 'death receptor’ pathway can
be monitored through the activity of the Fas death receptor as well as pro-caspase
8, a protein recruited to bind with Fas to form a death inducing signaling complex
(DISC), which can be visualized in Figure 1 (6). Other proteins such as the Bcl-2
family anti-apoptotic and pro-apoptotic proteins including Bcl-2 and Bax,
respectively, are considered key control points in the intrinsic or ‘mitochondria-
dependent’ pathway and can be used to gauge this pathway's activity as seen in
Figure 1 as well (5). Though the pathways are different, both the intrinsic and
extrinsic pathways have a similar end result, the release of cytochrome c from the
mitochondria.
It is hypothesized that our photodynamic therapy treatment induces an apoptotic
pathway as its mechanism of action to reduce cancer cell proliferation. To test if
our treatment is intrinsic or extrinsic various treatments known to induce a
specific pathway within a cell will be observed to make comparisons. Luteolin,
which is a natural flavonoid commonly found in fruits, vegetables, and medicinal
herbs, is one compound that has anticancer properties and was found to activate
both apoptotic pathways in MCF-7 cells (8). Another compound called genistein
is also a small, biologically active flavonoid that has been noted to inhibit cancer
cell proliferation by inducing the extrinsic apoptotic pathway (6). Previous studies
have also found that ethanol (EtOH) induces apoptosis using primarily the
intrinsic pathway (4).
Over time, the cancer cells can evolve and mutate proteins in these pathways to
become resistant to the perspective treatments like those mentioned above and to
chemo, radiation, and immune therapies offered (5). Thus it is crucial to find
newer, more effective treatment options that use different mechanisms of action
for treatment.
We are conducting experiments using a photodynamic therapy that activates a
photo isomerizing compound (PIC) and observing its effects on different cell
lines. Previous experiments have shown that light activation of the PIC in the
cell’s intracellular space resulted in membrane blebbing, an identifiable trait of
apoptosis, and a decrease in intracellular pH (pHi). Noting these changes in the
intracellular space, it is predicted that our therapy induces the intrinsic apoptosis
pathway. However, it is unknown as to which pathway is really induced.
Well Contents for Figure 2
1. MW protein ladder
2. Cytochrome c standard
3. Luteolin cell lysate
4. Genistein cell lysate
5. EtOH cell lysate
6. Photodynamic therapy
cell lysate
7. No treatment cell lysate
Detection of Intrinsic and Extrinsic Apoptosis
4-20% gradient
Figure 3. Compiled Western Blot
Results with Photodynamic Therapy
Figure 4. Compiled Western Blot
Results with Luteolin
Well Contents for
Figures 4 and 5
1. MW protein ladder
2. Cytochrome c standard
(~12 kDa & 70 kDa)
3. Fas (~45 kDa)
4. Procapase-8 (~55 kDa)
5. Bax (~21 kDa)
6. Bcl-2 (~22 & 25 kDa)
Figure 1. Intrinsic and extrinsic apoptotic pathways (7)
BaxPro-
caspase-8
Bcl-2
TRAIL - Rs
Fas/CD95
Cyt. C
DISC
luteolin + 24
hour incubation
period
genistein + 24
hour incubation
period
EtOH + 24 hour
incubation
period
photodynamic
therapy
Collection of Cell Lysates
Treated and non-treated cells + 1 hour
incubation with lysis buffer
Cell lysates + centrifuge to obtain proteins in
supernatant
Protein concentration determination using
Bradford assay
SDS-PAGE to detect apoptosis using cytochrome c
PVDF membrane
Western blotting with target antibodies;
stripping and reprobing between each antibody
β-actin, Bax and little/
no Bcl-2 antibody
detection with
chemiluminescence
Separation of Cell Lysate Proteins
Detection of Intrinsic and Extrinsic Apoptosis
β-actin, Fas, and
Procaspase-8 antibody
detection with
chemiluminescence
Intrinsic apoptosisExtrinsic apoptosis
Intrinsic apoptosisExtrinsic apoptosis Intrinsic apoptosisExtrinsic & intrinsic
apoptosis
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The UTSA College of Sciences
Science at the Forefront of
Basic and Translational Research
presents
October 9, 2015 | 8:00 am - 5:00 pm
UTSA Main Campus, HEB University Center
Oral and Poster Categories
• Chemistry and Biochemistry •
• Computer Science and Cyber Security •
• Infectious Diseases and Immunology •
• Mathematics and Statistics •
• Neurobiology •
• Physics & Astronomy and Nanotechnology •
• Regenerative and Molecular Medicine •
• Water, Energy, and Environment •
Keynote Speaker
Eliezer Masliah, M.D.
Professor of Neurosciences and Pathology
University of California, San Diego
Registration Now Open: http://www.utsa.edu/sciences/research_conference/
College of Sciences
Research Conference
Abstract Submission Deadline: September 20
8
Undergraduate
Awards
8
Graduate
Awards
I would like to thank the following:
• The Gdovin lab for all their thoughts and input on this proposed
experimental design
• and Dr. Gail Taylor
Supported by UTSA Collaborative Research Seed Grant Program Grant awarded to
MJG.
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