Investigating the Mechanism of Antioxidant MnTE-2-PyP as an SOD1 Mimetic in Prostate Cancer Progression

By: Joe GalaskePrincipal Investigator: Dr. Rebecca Oberley-DeeganUniversity of Nebraska Medical Center Department of Biochemistry and Molecular Biology

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.

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.

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.

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.

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.

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.

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.

T2E Inhibits Expression of Pro-Cancerous Genes, Including PAI-1

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T2E Inhibits Prostate Cancer Growth and Migration

MwhR.E., Crapo J.D. (2015). Mechanisms by which Mangansese Porphyrins AffectSignaling in Cancer Cells.

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.

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).

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).

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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.

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Normalized SOD1 Ex-pression (100 MOI)

Nuclear Extraction Assay (200 MOI)

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Normalized SOD1 Expression (200 MOI)

• However at 200 MOI large increases in both nuclear and cytosolic SOD1 levels are clearly evident.

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.

Control (No Virus) Empty SOD10

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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.

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.

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.