B. Kalyanaraman, Ph.D.

1
Doxorubicin-induced Cardiotoxicity and Antitumor
Action New Mechanistic Perspectives
B. Kalyanaraman, Ph.D. Department of
Biophysics Free Radical Research Center
SFRBM Lifetime Achievement Award Lecture Twelfth
Annual meeting of the Society for Free Radical
Biology and Medicine Austin, Texas November
16-20, 2005
2
Doxorubicin/Adriamycin
OH
O
COCH2OH
OH
C
D
B
A
O
OH
OCH3
O
O
H3C
NH2
OH
3
Broad-spectrum antitumor antibiotic Chronic
toxicity Induces cardiomyopathy or congestive
heart failure Post chemotherapeutic problems in
children Free radical mechanism of
cardiotoxicity Antioxidant and iron chelation
treatment Cardiomyocyte apoptosis Acute
toxicity Endothelial dysfunction
4
Redox-cycling of anthracycline quinones
O
Xanthine/ Xanthine Oxidase
C
NADH/ NADH Dehydrogenase
O
O2?-
H2O2
NADPH/ Cytochrome P-450 Reductase NOS
k 4.4?107M-1s-1
NADP
O?
O2
NAD
C
Uric Acid
O-
5
Kalyanaraman B, Perez-Reyes E, Mason
RP. Spin-trapping and direct electron spin
resonance investigations of the redox metabolism
of quinone anticancer drugs. Biochim Biophys Acta
630119-130, 1980
6
The EPR spectra of DOX semiquinone
and DMPO-superoxide temporal relationship
DOX-semiquinone
DMPO- superoxide
Kalyanaraman B, Perez-Reyes E, Mason RP. Biochim
Biophys Acta 630119-130, 1980
7
Role of iron in DOX-induced oxidative damage
O
C
H2O2
NAD(P)H H
O
IRON
NAD(P)
O?
O2
Hydroxyl radical
C
O-
Oxidative stress
8
Chelated iron-catalyzed Haber-Weiss mechanism
Radical-driven Fenton Reaction
Kalyanaraman B, Morehouse KM, Mason RP. Arch
Biochem Biophys 286164-170, 1991
9
Kalyanaraman B, Sealy RC, Sinha BK. An electron
spin resonance study of the reduction of
peroxides by anthracycline semiquinones. Biochim
Biophys Acta 799270-275, 1984
10
Time-lag in semiquinone formation during
anaerobic reduction of adriamycin by
xanthine/xanthine oxidase The effect of H2O2
and chelators
- H2O2
H2O2, no chelator
H2O2, EDTA
H2O2, DTPA
H2O2, DFO
Kalyanaraman B, Morehouse KM, Mason RP. Arch
Biochem Biophys 286164-170, 1991
11
EPR spectra of DMPO radical adducts
observed during the lag period
DMPO-OH
DMPO-CH (CH3) OH
Kalyanaraman B, Morehouse KM, Mason RP. Arch
Biochem Biophys 286164-170, 1991
12
Redox-cycling of the iron-sulfur cluster in
aconitase
Fridovich I. J Biol Chem 27218515, 1997 Gardner
PR. Biosci Res 1733, 1997 Vasquez-Vivar J, et
al. J Biol Chem 27514064, 2000
13
THE FREE RADICAL HYPOTHESIS OF CARDIOTOXICITY
O2
DOX._
NAD(P)H
F
FH.
H2O2
DOX
FH2
NAD(P)
Fe-S clusters
H2O2
.OH Fe (IV) O DOX. Fe
Fe2
mitochondrial lipid peroxidation
mitochondrial protein oxidation
mitochondrial DNA damage
sarcolemmal, mitochondrial and sarcoplasmic
reticular changes
Contractile Impairment
Minotti et al. FASEB J. 13199, 1999
14
Vasquez-Vivar J, Martasek P, Hogg N, Masters BSS,
Pritchard KA, Kalyanaraman B. Endothelial nitric
oxide synthase-dependent superoxide generation
from adriamycin. Biochemistry 3611293-11297, 1997
15
Reductive activation of adriamycin by eNOS
H2O2
ONOO-
O2?-
O2
L-citrulline
NO?
Adr
Adr ?-
L-Arg
BH4
FMN
HEME
NADP
FAD
NADPH
NOS ACTIVITY
NADPH OXIDASE ACTIVITY
16
Adriamycin-dependent inhibition of eNOS activity
Biochemistry 3611293-11297, 1997
17
Duquaine D, Hirsch GA, Chakrabarti A, Han Z,
Kehrer C, Brook R, Joseph J, Schott A,
Kalyanaraman B, Vasquez-Vivar J, Rajagopalan
S. Rapid-onset endothelial dysfunction with
adriamycin Evidence for a dysfunctional nitric
oxide synthase. Vasc Med 8101-107, 2003
18
Sawyer DB, Fukazawa R, Arstall MA, Kelly
RA. Daunorubicin-induced apoptosis in rat cardiac
myocytes is inhibited by dexrazoxane. Circ Res
84257-265, 1999
19
Apoptotic signaling pathways
Fas/TNF2-receptor
Fadd
Bid
Bad
Caspase-8
Bcl-2
Bcl-2
Bcl-2
Bax
Bax
Bad
Bcl-2
p53
mitochondria
intrinsic Pathway
extrinsic Pathway
Bax
Nucleus
Bax
Cytochrome c release
caspase-3, 6, 7
effector
caspases
cytochrome c
caspase-9
Apaf
-
apoptosis
procaspase-9
-
apoptosome
20
Doxorubicin-induced apoptosis in BAEC as shown by
TUNEL
A
B
C
D
E
F
G
100
80
60
apoptosis
40
20
0
A
B
C
D
E
F
G
1.0 µM DOX 100 µM MnTBAP
1.0 µM DOX 100 µM PBN
1.0 µM DOX 50 µM Ebselen
J Biol Chem 27533585-92, 2000
21
Effect of DOX on caspase-3 activation in
cardiomyocytes and endothelial cells
Myocytes
BAEC
70
200
60
160
50
120
40
nmoles pNA/mg protein
nmoles pNA/mg protein
30
80
20
40
10
0
0
0
1
2
4
8
16
24
0
1
2
4
6
8
Time (h)
Time (h)
22
Effect of iron chelators on DOX-induced caspase-3
proteolytic activation
Cells preincubated with iron chelators for 2h
Cells were not preincubated with iron chelators
70
60
50
40
caspase-3 like activity (nmole pNA/ mg protein)
30
20
10
0
DOX
HBED
Control
desferral
ICRF-187
DOX HBED
DOX HBED
DOX desferral
DOX ICRF-187
DOX desferral
DOX ICRF-187
J Biol Chem 27717179-87, 2002
23
Regulation of iron uptake by IRP/IRE
Klausner RD et al., Cell 7219-28, 1993
24
Effect of DOX on aconitase and IRP-1 activities
in endothelial cells
0.4
2-ME
- 2-ME
IRE / IRP
0.3
Aconitase activity (U/ mg protein)
0.2
free probe
0.1
0
1
2
4
8
0
1
2
4
8
Time (h)
Time (h)
0
0
2
4
6
8
16
Time (h)
J Biol Chem 27717179-87, 2002
25
Effect of DOX on transferrin receptor levels in
endothelial cells
350
300
TfR
250
(95 kDa)
200
of control
0
2
4
6
8
16
150
Time (h)
100
50
0
0
2
4
6
8
16
Time (h)
J Biol Chem 27717179-87, 2002
26
Effect of doxorubicin on 55Fe uptake in BAEC
20000
DOX
medium
35000
25000
cell lysate
30000
15000
20000
25000
55Fe uptake (cpm/ mg protein)
15000
cpm/ ml medium
cpm/ mg protein
20000
- DOX
10000
15000
10000
10000
5000
5000
5000
0
0
0
0
2
4
6
8
16
DOX
DOX
Control
Control
Time (h)
J Biol Chem 27717179-87, 2002
27
Effect of anti-transferrin receptor antibody on
DOX-induced 55Fe uptake in BAEC
20000
15000
55Fe uptake (cpm/ mg protein)
10000
5000
0
DOX 55Fe (0 h)
DOX 55Fe (2 h)
DOX 55Fe TfR ab (2 h)
DOX 55Fe TfR ab (6 h)
J Biol Chem 27717179-87, 2002
28
Effect of anti-TfR antibody and iron chelators on
DOX- induced oxidative dtress, as measured by DCF
staining
e
d
c
b
a
0.5 µM DOX
2 µM DOX
anti-TfR ab alone
Control
0.5 µM DOX anti-TfR ab
1000
800
g
h
f
i
DCF intensity (arbitrary units)
600
400
200
2 µM DOX anti-TfR ab
0.5 µM DOX desferral
0.5 µM DOX HBED
0.5 µM DOX ICRF-187
0
g
a
b
c
e
f
h
i
d
J Biol Chem 27717179-87, 2002
29
Effect of anti-transferrin receptor antibody
on DOX-induced on caspase-3 proteolytic activation
90
DOX
80
DOX anti-Tfr ab
70
60
caspase-3 like activity (nmole pNA/ mg protein)
50
40
30
20
10
0
0
2
4
6
8
16
Time (h)
J Biol Chem 27717179-87, 2002
30
A proposed model for oxidant-induced cellular
uptake of iron
H2O2
J Biol Chem 27717179-87, 2002
31
Wang S, Konorev EA, Kotamraju S, Joseph J,
Kalivendi S, Kalyanaraman B. Doxorubicin induces
apoptosis in normal and tumor cells via
distinctly different mechanisms. J Biol Chem
27925535-25543, 2004
32
Effect of GPx-1 overexpression on DOX-induced p53
activation and apoptosis
100
100

80
80

60
60
of apoptosis

of apoptosis



40
40
20

20



0
0
control
0.1
0.5
1
control
0.1
0.5
1
BAEC
DOX (?M)
DOX (?M)
PA-1
10
10

with GPx-1

8
without GPx-1
8

6
6
Caspase-3 activity
(increasing fold)
Caspase-3 activity
(increasing fold)
4
4

2
2
0
0
control
DOX
control
DOX
BAEC
PA-1
J Biol Chem 27925535-25543, 2004
33
Yen H-C, Oberley TD, Vichitbandha S, Ho Y-S, St.
Clair DK. The protective role of manganese
superoxide dismutase against adriamycin-induced
acute cardiac toxicity in transgenic mice. J Clin
Invest 981253-1260, 1996
34
Hu Y, Rosen DG, Zhou Y, Feng L, Yang G, Liu J,
Huang P. Mitochondrial MnSOD expression in
ovarian cancer Role in cell proliferation and
response to oxidative stress. J Biol Chem (in
press, 2005)
35
Mitochondrially-targeted antioxidants
36
DOX-induced NFAT translocation, FasL
expression, and apoptosis
Kalivendi S, et al. Biochem J 389527-539, 2005
37
Effect of antioxidant enzymes on
DOX-induced apoptosis and NFAT activation
DOX
Control
DOX MnSOD
DOX GPx1
DOX Ad-LacZ
Biochem J 389527-539, 2005
38
Mitochondria, calcium, and NFAT in DOX-induced
apoptosis in cardiac cells
DOX
Reductase /dehydrogenase
DOX DOX
Fas L Ab
DPI
O2
O2
H2O2?
Mito-Q
FasL
Ca2
Fas
?
Calcineurin?
Ca2 ?
CsA
FasL mRNA?
BAPTA
Cyt C release
C8i
-NFAT
P
NFAT
Caspase 8?
NFAT-I
Caspase 3?
C3i
APOPTOSIS
39
Acknowledgments

• Srigiridhar Kotamraju, Ph.D.
• Shasi Kalivendi, Ph.D.
• Eugene Konorev, Ph.D.
• Joy Joseph, Ph.D.
• Deepika Aggarwal, Ph.D.
• Ray Migrino, M.D.

40
EPR spectra of the semiquinone free radical
derived from daunomycin on reduction with
xanthine/xanthine oxidase
Schreiber J, Mottley C, Sinha BK, Kalyanaraman B,
Mason RP. J Am Chem Soc 109348-351, 1987