Effect of Amifostine On Effect of Amifostine On Mitochondrial Superoxide Mitochondrial Superoxide Dismutase Expression In Dismutase Expression In

INS-1 Beta CellsINS-1 Beta Cells

Daniel Turner-LloverasDaniel Turner-Lloveras

Dr. Michael RoeDr. Michael Roe

Type 2 Diabetes Type 2 Diabetes MellitusMellitus

• Insulin ResistanceInsulin Resistance• Beta Cell DysfunctionBeta Cell Dysfunction

• HyperglycemiaHyperglycemia• Oxidative Stress (ROS)Oxidative Stress (ROS)• Beta Cell DegradationBeta Cell Degradation• Beta Cell DeathBeta Cell Death• Complications of DiabetesComplications of Diabetes

• Blindness, Heart Disease, Kidney Problems, etc.Blindness, Heart Disease, Kidney Problems, etc.

O2- H2O2

MnSOD

H2O O2

Catalase

H2O

Glutathione Peroxidase

Endogenous Antioxidant Endogenous Antioxidant PathwaysPathways

normal [glucose]

MnSOD(?)

Oxidative Damage

H2O O2

HO--

H2O

Glutathione Peroxidase

Enhanced Endogenous Enhanced Endogenous Antioxidant Defense during Antioxidant Defense during

hyperglycemiahyperglycemia??

O2- H2O2

Catalase(?)

HIGH [GLUCOSE]

(high mitochondrialmetabolic activity)

HypothesisHypothesis

• Amifostine induces upregulation of Amifostine induces upregulation of MnSOD (Sod2) via activation of NF-MnSOD (Sod2) via activation of NF-κβκβ

AmifostineAmifostine

• A phosphorothioate that is A phosphorothioate that is metabolized to the free thiol metabolized to the free thiol metabolite WR-1065.metabolite WR-1065.

• Developed by military to protect Developed by military to protect soldiers during nuclear warfare.soldiers during nuclear warfare.

• The only drug approved by the The only drug approved by the FDA specifically as a FDA specifically as a radioprotector.radioprotector.

Amifostine ↑ NF-κβ ↑ MnSOD

MnSOD(?)

Oxidative Damage

H2O O2

HO--

H2O

Glutathione PeroxidaseO2- H2O2

Catalase(?)

HIGH [GLUCOSE]

METHODSMETHODS

• INS-1 Rat Insulinoma Cell LineINS-1 Rat Insulinoma Cell Line• 40 µM WR-1065 40 µM WR-1065 • Real-Time/ Quantitative PCR Real-Time/ Quantitative PCR

(QPCR)(QPCR)• Western ImmunoblottingWestern Immunoblotting

0 4 8 24 48

30 min

Treatment Hours

QPCRQPCR

MnSod Time Course

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35 40 45 50

Treatment

Fold

Change

Untreated

Treated

MnSod Gene Expression

0

0.5

1

1.5

2

2.5

3

3.5

4

0 hour 4 hours 8 hours 24 hours 48 hours

Treatment

Fold

Change

Untreated

Treated

QPCR DATAQPCR DATA

• Drug induced transient increase in Drug induced transient increase in MnSOD gene expressionMnSOD gene expression

• No data for Glutathione Peroxidase No data for Glutathione Peroxidase and Catalaseand Catalase

MW 0 -- 4 + -- 8 + -- 24 + -- 48 +

-- = Control

+ = Treated with WR-1065

MnSOD

β-Actin

Western Western ImmunoblottingImmunoblotting

Western Blot DATAWestern Blot DATA

• Transient Increase in MnSOD levels Transient Increase in MnSOD levels in both drug treated and controlin both drug treated and control

CONCLUSIONCONCLUSION

• Amifostine upregulates Amifostine upregulates sod2sod2 (MnSOD) gene expression.(MnSOD) gene expression.

• Effects on MnSOD protein levels Effects on MnSOD protein levels are uncertain.are uncertain.

Future DirectionsFuture Directions

• Refine Technique and repeat Refine Technique and repeat experimentsexperiments

• Investigate role of NF-Investigate role of NF-κκBB• Mitochondrial function and insulin Mitochondrial function and insulin

secretionsecretion• In vivoIn vivo experiments with Fatty experiments with Fatty

Zucker Diabetic RatsZucker Diabetic Rats

AcknowledgementsAcknowledgements

• Dr. Michael RoeDr. Michael Roe• Dr. David GrdinaDr. David Grdina• Dr. Jeff MurleyDr. Jeff Murley• Luis LandaLuis Landa• Dr. Mark HarbeckDr. Mark Harbeck• Kelly KaiharaKelly Kaihara• Dr. Kajorn Kitiphongspattana Dr. Kajorn Kitiphongspattana • Dr. Vernon TowleDr. Vernon Towle• Dr. Eugene ChangDr. Eugene Chang