Adjuvant Vitamin E
and Ovarian Cancer
Alysha Hartman
Mentor: Debbie Mustacich, Ph.D.
Linus Pauling Institute
Oregon State University
Outline
Ø Introduction
•  Ovarian Cancer
•  Cisplatin
•  Vitamin E
Ø Question
Ø Hypothesis
Ø Experimental Design
Ø Results
Ø Summary
Ø Future Studies
F344 Female Rats
Ovarian Cancer
Ø 5th leading cause of cancer-related deaths in women
in the U.S.
•  Accounts for about 6% of deaths
Ø Highest mortality rate of gynecologic cancers
Ø Most patients have widespread disease at diagnosis
Cisplatin (CDDP)
Ø Platinum containing chemotherapeutic drug
Ø Highly active against a number of cancers:
•  Ovarian, Lung, Cervical, Head & Neck, Testicular
Ø Side effects include:
•  Nephrotoxicity, ototoxicity, and neurotoxicity
CDDP-Induced Neuropathy (CIPN)
Ø Affects the nerves that carry sensations to the brain
Ø Major dose-limiting adverse side effect of CDDP
Ø Symptoms include:
•
Tingling & burning in hands and feet
•
Tremors
•
Numbness
CDDP-induced Neuropathy & Vitamin E
Ø Mechanism of CIPN is undetermined.
•  Platinum accumulation
Ø Clinical and histologic features are similar to
those seen in Vitamin E deficiency neuropathy.
(Muller ‘83, Sokol ’88)
Ø CDDP decreases plasma Vitamin E in humans.
(Weigl ’98)
Vitamin E (RRR-α-Tocopherol)
Ø RRR-α-Tocopherol (α-T): natural form of vitamin
E preferentially retained in the body. (Traber ’05)
Antioxidant role of α-T
Ø Lipid soluble antioxidant
•  Found in cell membranes
Ø Protects cellular lipids from oxidation
•  lipid peroxidation
Ø Biomarkers of Lipid peroxidation:
•  F2-Isoprostanes
•  Malondialdehyde (MDA)
Central Question
Can the neurotoxicity of CDDP be mitigated to
allow increased survival without decreased
quality of life for ovarian cancer patients?
Central Hypothesis:
1)  CDDP depletes tissue α-T by an oxidative stress
mechanism leading to neurotoxicity
2)  Adjunct α-T will prevent CDDP-mediated α-T
depletion, thereby preventing neurologic damage
3)  Adjuvant α-T will not decrease CDDP anti-tumor
efficacy
Pre-clinical Model of Ovarian
Cancer
Vitamin E (α-T) and Cisplatin (CDDP) Treatment Schedule
Days:
Week 1
Week 4
(Sun, Tue,
Thur)
GROUP
Week 5
(Friday)
Weeks 5-8
(Mon &
Thur)
Weeks 5-8
(Tues &
Fri)
Week 9
TREATMENT
A Saline
IP inject
cells
SC saline
SC saline
SC saline
SC saline
Sacrifice
B α-T
IP inject
cells
SC α-T
SC saline
SC α-T
SC saline
Sacrifice
C Cisplatin
IP inject
cells
SC saline
SC saline
IP CDDP
SC saline
SC saline
IP CDDP
Sacrifice
D α-T/
Cisplatin
IP inject
cells
SC α-T
SC saline
IP CDDP
SC α-T
SC saline
IP CDDP
Sacrifice
* Rats treated with CDDP receive an accumulative dose of 18mg / kg
Adjuvant α-T does not alter CDDPinduced weight loss
Adjuvant α-T increases CDDP
Anti-tumor Efficacy
Saline
CDDP
CDDP and α-T
Adjuvant α-T decreases Tumor
Incidence and Multiplicity
Number of Rats Per Group
Tumors
Per Rat
0
<10
10-50
50-100
100+
2/8
6/8
1/12
8/12
3/12
Group A
Group B
Group C
2/16
2/16
1/16
8/16
3/16
Group D
10/16
2/16
2/16
1/16
1/16
Adjuvant α-T prevents tumor- and CDDPinduced depletion of α-T
αT
P
D
+
D
C
D
D
P
C
αT
lin
e
+
P
D
D
C
αT
P
D
+
D
D
D
P
C
C
αT
lin
e
Sa
ol
on
tr
+
P
D
D
C
C
αT
P
D
D
C
αT
Sa
lin
e
ol
αT
P
D
D
#
40
0
0
on
tr
C
D
D
αT
80
C
C
#
lin
e
*#
#
600
400
200
Sa
ol
α−T (nmol/g)
on
tr
800
C
+
P
C
C
C
α−T (nmol/g)
0
#
400
0
Tumors
*#
*
*
*#
20
Adrenal
1400
1200
1000
*
40
*#
αT
P
D
D
lin
e
ol
on
tr
C
+
αT
40
αT
0
80
Sa
* # *#
D
C
D
D
αT
lin
e
Sa
C
on
tr
ol
0
*
40
*#
*#
ol
40
600
400
200
120
on
tr
60
*#
*#
*#
Plasma
80
Sa
80
600
400
200
α−T (nmol/g)
*#
α−T (nmol/g)
600
400
200
Lung
Kidney
*#
P
D
α−T (µM)
P
α−T (nmol/g)
Liver
* = p<0.05 vs. non-tumor controls; # = p<0.05 vs. saline treated tumor-bearing rats
Adjuvant α-T improves spinal cord
α-T levels compared to CDDP alone
Cervical
40
*#
30
*
20
Thoracic
50
*#
*
10
α−T (nmol/g)
0
40
30
*
*
*
*
20
10
40
*
30
*
20
10
Cauda Equina
40
30
#
*
#
*
20
10
αT
P
D
+
P
D
D
αT
D
C
C
+
P
D
D
C
C
αT
P
D
D
C
αT
Sa
lin
e
ol
on
tr
on
tr
ol
Sa
lin
e
0
0
C
αT
D
P
C
D
50
α−T (nmol/g)
α−T (nmol/g)
C
C
Lumbar
50
+
D
D
P
αT
D
D
C
P
C
+
on
tr
ol
αT
P
D
D
αT
lin
e
Sa
C
on
tr
ol
0
Sa
lin
e
α−T (nmol/g)
50
* = p<0.05 compared to non-tumor controls; # = p<0.05 compared to saline treated tumorbearing rats.
600
*
400
*
*#
*#
200
αT
P
D
+
D
D
D
P
C
C
αT
lin
e
Sa
on
tr
ol
0
C
+
5-Series F2-Isoprostane
(pg/mL)
αT
P
P
D
D
C
αT
D
D
C
+
P
D
D
C
0
lin
e
αT
P
D
D
C
αT
lin
e
Sa
C
on
tr
ol
0
100
Sa
100
*#
*#
ol
*#
*#
*
200
on
tr
200
*
C
*
300
(pg/mL)
*
15S-8-iso-PGF2α
300
(pg/mL)
15R-8-iso-PGF2α
Adjuvant α-T prevents tumor- and
CDDP-induced elevation of plasma
F2-Isoprostanes
* = p<0.05 compared to non-tumor controls; # = p<0.05 compared to saline treated tumorbearing rats.
* = p<0.05 compared CDDP alone
T
C
D C
DD
PD
+P+
α- α
T -
P
D
D
C
p = 0.07
T
80.10
6
40.05
2
00.00
-αT-
+α
C CD
D D
D P
P +
+ α
α- -T
T
CC
DD
DD
PP
*
200.15
P+
6
0.05
4
2
0.00
0
0.20
25
DC
DD
PD
0.10
8
Tumor
Cauda Equina
C
*
0.15
20
P = 0.07
30
Platinum (µg/mg)
25
Platinum (µg/mg)
0.20
0.10
8
60.05
4
20.00
0
C DP
D
D
P
Lung
Lumbar
30
*
DC
PD
C CD
D D
D P
P +
+ α
α- -T
T
0.00
0.15
20
D
0.05
10
25
C
0.10
Thoracic
Liver
300.20
Platinum (µg/mg)
20
Platinum (µg/mg)
*
0.15
0
Platinum (µg/mg)
Platinum
(µg/mg)
Cervical
Kidney
0.20
30
C C
D D
D D
P P
Platinum(µg/mg)
(µg/mg)
Platinum
Adjuvant α-T reduces spinal cord and
lung platinum but not tumor platinum
Summary
α-T plus CDDP:
Ø  Increases CDDP anti-tumor efficacy
•
Decreased tumor burden
Ø  Prevents CDDP-induced decreases in tissue and plasma
α-T
Ø  Prevents tumor- and CDDP-induced lipid peroxidation
Ø  Decreases accumulation of platinum in spinal cord
tissues but not tumors
Ongoing & Future Studies
Ongoing Studies:
Ø Tissue analysis
•  MDA – oxidative stress
•  Glutathione – antioxidant
Ø Histology
•  proliferation markers
Future Work:
Ø Adjuvant Vitamin C
Ø Clinical Trials
Acknowledgements
Ø Debbie Mustacich, Ph.D.
Ø  Kevin Ahern, Ph.D.
Ø Shannon Pease
Ø  ACS Research Scholar
Ø Michelle Beehler
grant 120548-
Ø Devin Corrigan
RSG-11-075-01-CCE
Ø Giovanna Coto, M.A., DVM
Ø Christiane Löhr, DVM, Ph.D.
Ø F344 Rats
Ø Linus Pauling Institute
Ø URISC
Questions?
Pilot Study in Healthy Rats: CDDP
Decreases Ganglia α-T **
α−T (nmol/g)
50
Veh
Vit E
CDDP
CDDP + VE
b
40
a,b
a
30
b
d
a
c
c
20
10
0
D. Ganglia
V. Ganglia
Ganglia
** Columns within the same tissue but with different letter designations are significantly different, p<
0.05
Lipid Peroxidation
1)  Initiation
•  Production of a carbon radical
2)  Propagation
•  Peroxyl radical and an additional
carbon radical are formed
3)  Termination
•  Two peroxyl radicals combine to
stop the chain reaction
F2-Isoprostane from Arachidonic Acid