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Characterization of Apoptotic Pathways Induced in Novel Photodynamic Cancer TreatmentKendra Hanslik, Justin Avila, Jennifer Watts, Matthew Gdovin

Department of Biology, The University of Texas at San Antonio, San Antonio, TX. 78249

AbstractCurrent 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 HypothesisThe 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

DIS

C

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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