The following project was conducted by myself with the assistance of Rebecca E. Oberley-Deegan, Ph.D., Elizabeth A. Kosmacek, Ph.D., and Qiang Tong, Ph.D. This presentation talks about my summer research (2015) at the University of Nebraska Medical Center and details the background, methods, and results of my project.

1. Investigating the
Mechanism of Antioxidant
MnTE-2-PyP as an SOD1
Mimetic in Prostate Cancer
Progression
By: Joe Galaske
Principal Investigator: Dr. Rebecca Oberley-Deegan
University of Nebraska Medical Center Department of Biochemistry and
Molecular Biology

2. Prostate Cancer
• Prostate Cancer is the most common cancer among men
• 1 in 7 men will be diagnosed with prostate cancer during his
lifetime.
• Prostate cancer is second leading cause of cancer death
among men, behind lung cancer.
• While serious, most men diagnosed with prostate cancer do
not die from it.
• Roughly 3 million men diagnosed with prostate cancer are still
alive today.

3. Effects of Radiation Treatment
• To treat prostate cancer, radiation therapy is routinely used as a
treatment method.
• Although radiation therapy effectively kills prostate tumor cells,
surrounding tissue is also damaged.
• Long-term complications from radiation therapy include:
• Bowel and rectal wall damage
• Lower urinary tract symptoms (urgency and frequency)
• Erectile dysfunction
• Urethral stricture
• Incontinence
• Damage of normal tissue is usually progressive and irreversible.

4. Role of ROS in Prostate Cancer
• Many studies have implicated free radicals (oxidative stress) as
a cause of radiation-induced tissue damage and chronic
inflammation.
• Specifically reactive oxygen species (ROS) have been shown to
drive complications, such as erectile dysfunction, post-irradiation.
• Radiation can cause water radiolysis that directly produces
hydroxyl radical or forms superoxide.
• This enhancement of ROS promotes cancer cell death, but has
two main drawbacks:
• Cancer resistance to higher ROS levels and aggressive regrowth.
• Enhanced ROS also damages and kills normal cells, causing the
previously discussed complications.

5. Oxidative Environment is Necessary
for Cancer Progression
• An oxidative environment is also critical to cancer cell
signaling, growth, and invasive capabilities.
• Previous studies have shown that cancer cells overexpress
NADPH oxidases (NOX) enzymes, which generate superoxide.
• Increased superoxide and hydrogen peroxide levels have been
shown to promote cancer progression via altering cell
signaling pathways.
• Likewise, increased levels of ROS also allow cancer cells to
alter the extracellular environment, promoting cancer growth
and invasion.

7. Superoxide Dismutase (SOD)
• Superoxide dismutase (SOD) enzymes are naturally occurring
enzymes which scavenge superoxide to generate hydrogen peroxide.
• SOD exists in three forms:
• SOD1 (CuZnSOD) (Only SOD found in nucleus, as well as cytoplasm)
• SOD2 (MnSOD) (Mitochondria)
• SOD3 (EcSOD) (Extracellular matrix)
• SOD is down-regulated in many primary cancers.
• Reducing the oxidative environment via overexpression of SOD in
cancer cell results in a more normal phenotype.
• Therefore antioxidants, molecules that scavenge free radicals, when
used in combination with radiation therapy should minimize injury
to normal tissue.

8. MnTE-2-PyP (T2E)
• T2E is a potent, small molecular weight antioxidant that
scavenges multiple free radicals including superoxide, lipid
peroxides and peroxynitrite.
• T2E is believed to affect a wide variety of disease states
through its ability to alter cell signaling pathways.
• One way T2E reduces inflammation is by inhibiting NF-κB signaling by
altering the surrounding redox environment.
• Previous studies have shown T2E to be found in
concentrations around 3x higher in the nucleus as compared
to the cytoplasm.
Makinde A.Y., Luo-Owen X., Rizvi A., Gridley D.S., et. al. (2009). Effect of a Metalloporphyrin Antioxidant (MnTE-2-PyP) on the
Response of a Mouse Prostate Cancer Model to Radiation. Anticancer Research (29): 107-118.

9. T2E Inhibits Cancer Progression
• Many studies have demonstrated multiple mechanisms by
which T2E inhibits cancer growth:
• T2E hinders cancer cell proliferation by restricting aerobic
glycolysis.
• T2E inhibits HIF-1α activity, thereby limiting angiogenesis.
• T2E has been shown to inhibit expression of pro-cancerous genes
PAI-1, TGF-β, and FGF-1.
• T2E has also recently been shown to inhibit HAT enzyme, p300,
which serves as an enhancer for transcription factors such as HIF-
1α and NF-κB.
• Therefore, it is presumed that T2E alters the redox
environment by serving as an SOD mimetic.

11. T2E Inhibits Expression of Pro-
Cancerous Genes, Including PAI-1
0.0
0.5
1.0
1.5
FoldchargeofRNAlevel
(normalizedby18srRNA)
P =0.0047
P =0.016
P =0.014
C on T2E C on T2E
0G y 20G y
C on T2E C on T2E
0G y 20G y
C on T2E C on T2E
0G y 20G y
T G F - 2 PA I-1 FG F -1

12. T2E Inhibits Prostate Cancer
Growth and Migration
MwhR.E., Crapo J.D. (2015). Mechanisms by which Mangansese Porphyrins Affect
Signaling in Cancer Cells.

13. My Project
• Rationale:
• Transfected SOD1 will be found in the nucleus, as well as the
cytoplasm.
• Overexpression of SOD1 will result in decreased expression of
pro-cancerous gene PAI-1.
• Overexpression of SOD1 (CuZn) via adenovirus transfection will
limit cancer cell growth in vitro.

14. 72 Hours as an Optimal Time
• To determine optimal time of collection, a time course
experiment using 10 and 100 MOI was ran.
• Western blot showed clearly elevated expression of SOD1 at 72
hours (100 MOI).

15. SOD1 Activity at 72 Hours
• To demonstrate activity of transfected SOD1, a protein activity
assay was used.
• After repeated attempts, clearly elevated SOD1 activity was
recorded at 72 hours (100 MOI).
0
50000
100000
150000
200000
250000
300000
No 48 Empty
48
SOD1
48
No 72 Empty
72
SOD1
72
SOD1 Activity Assay
No
48
E
48
SOD1
48
No
72
E
72
SOD1
72

16. Nuclear Extraction Assay (100 MOI)
• To determine nuclear vs. cytosolic SOD1 concentrations, a
nuclear extraction assay was used.
• At 100 MOI, no significant increase in nuclear SOD1 levels were
recorded.
0
0.01
0.02
0.03
0.04
0.05
No 72
Cyto
Empty
72 Cyto SOD1 72
Cyto
No 72
Nuclear
Empty
72
Nuclear
SOD1
72
Nuclear
Normalized SOD1
Expression (100 MOI)

17. Nuclear Extraction Assay (200 MOI)
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
No 72
Cyto
Empty
72 Cyto SOD1 72
Cyto
No 72
Nuclear
Empty
72 Nuclear
SOD1 72
Nuclear
Normalized SOD1 Expression
(200 MOI)
• However at 200 MOI large increases in both nuclear
and cytosolic SOD1 levels are clearly evident.

18. Overexpression of SOD1 Inhibits
Cancer Cell Growth
• After determining optimal time, MOI, SOD1 activity, and
nuclear concentrations, a clonogenic (cell growth) assay is
currently being ran to demonstrate how elevated levels of
SOD1 effects cancer cell survival.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Control (No Virus) Empty SOD1
%Survival
100 MOI

19. Future Directions
• Future experiments can be completed that replicates and
confirms data.
• I am planning on next performing qt-PCR to look at expression
of pro-cancerous gene PAI-1 after overexpression of SOD1.
• Similar experiments with transfected catalase, and effects on
cancer cell survival.
• ChIP assays to look at the effect of overexpression of SOD1 on
binding of transcription factors HIF1-α, NF-κB, p300, etc.

20. Conclusions
• My experiment has thus far demonstrated the mechanism of
T2E as an SOD1 mimetic, by showing that elevated SOD1
levels produce similar effects to T2E treatment.
• Targeting the oxidative environment of cancer cells provides
an effective means to inhibit cancer progression without
causing damage to normal tissue cells in humans.

21. Thank You
• A special thank you to Dr. Rebecca Oberley-Deegan, Elizabeth
(Annie) Kosmacek, and Qiang Tong for their time/help with my
project.
• Additional thanks to Brandon Griess for his help and advice on
my SOD1 activity assay.
• A final thank you to the University of Nebraska Medical Center
Department of Biochemistry and Molecular Biology for
allowing me to take part in the SURP program.
• National Institutes of Health Grants 1R01CA178888.