ß-Lapachone, a ‘Kiss of death’ therapy for cancer
David A. Boothman, Ph.D.
Laboratory of Molecular Stress Responses
Program in Cell Stress and Cancer Nanomedicine
Simmons Comprehensive Cancer Center
University of Texas Southwestern Medical
Center at Dallas
September 14, 2008
Cellular Damage
IR
New Gene Expression
xips
Exploitable Target: Expressed
in Human Tumors, not (or low)
in Normal Tissues
NQO1
Bioactivate Drug For
Tumor-Selective Killing
ß-Lapachone
-Not cell cycle regulated
-Not affected by drug resistance
-Not dependent on p53 status
-Not affected by hypoxia
-Not dependent on caspases
-Selective for tumors
β-Lapachone (ß-Lap)
O
O
O
CH 3 CH 3
The Lapacho Tree
Tabebuia avellanedae
Repair
Surviving Fraction
ß-Lapachone radiosensitized human cancer, but not normal, cells
3 Gy
-IR
(0 Gy)
+IR
(3 Gy)
2
4
6
8
10
Control
12 14
ß-Lapachone (µM)
Radiosensitization Mechanism:
PreDuring Post- ß-Lap (5 µM, 4h)
Treatment Condition
IR induces NQO1(xip3), the principal determinant of ß-lap
cytotoxicity (Pink, et al., JBC, 2000).
28S
xip3
18S
Boothman et al., Cancer Res., 1989
Boothman et al., PNAS, 1990. 1993
TIM
E(hrs)POST-IR(450cGy)
NQO1: An Important Target for Cancer Therapy
-Early
marker
of
carcinogenes
-Up-regulated after carcinogenic cell stres
-Over-expressed in many cancers, as well a
in angiogenic endothelial cells
Tumor-selective NQO1 Elevation
• 80% Breast Cancers, 10- to 20-fold **
• 70% Prostate Cancers, 10- to 20-fold **
• 60% Colon Cancers, 5- to 10-fold
• 90% Pancreatic Cancers,
(J. Cullen, U. Iowa)
• 70% NSCLC (not SCLC), 20- to 40-fold **
** Will discuss isogenic models +NQO1
NQO1 Is Elevated In Nonsmall Cell Lung Cancer (NSCLC)
NQO1 Expression Confers Cytotoxicity to
H596 NSCLC Cells
NQO1
α-tubulin
NQO1 Enzyme Activity
(nmol cyt C red/min/prot)
Bey et al.,
PNAS, 2007
NQO1-dependent lethality in human prostate cancer cells
LN-pcDNA3
ß-Lapachone
LN-pcDNA3 + Dic
100
30
25
20
15
10
5
0
Relative Survival
NQO1 Activity
(X 100)
LN-NQ Cl 1
LN-NQ Cl 1 + Dic
LN-NQ Cl 2
10
LN-NQ Cl 2 + Dic
LN-NQ Cl 3
LN-NQ Cl 3 + Dic
1
LN-NQ Cl 4
LN-NQ Cl 4 + Dic
LN-NQ Cl 10
0.1
0
2
4
6
8
10
12
LN-NQ Cl 10 + Dic
ß-lap (µM)
100
LN-NQ Cl 10
Relative Survival
Menadione
pcDNA3
Conclusion
NQO1 “bioactivates” ß-Lap
10
NQO1 “inactivates” Menadione
1
0
5
10
15
20
25
Menadione (µM) Planchon et. al., Exp Cell Res., 2001
NQO1-Mediated Reduction of β-Lap and Menadione
NAD(P)H NAD(P)+
NQO1
Menadione (HQ)
Stable
Menadione (Q)
Stable Reduction
NAD(P)H NAD(P)+
NQO1
β-Lapachone (Q)
β-Lapachone (HQ)
Unstable
β-Lap (SQ•)
Futile Cycle
β-Lapachone Redox Cycling
O
NAD(P)H
NAD(P)+
OH
Dicumarol
O
OH
NQO1
O
O
NAD(P)+
H3C
NAD(P)H
CH3
β-Lapachone

O2
H3C
P450R
b5R NAD(P)H
Hydroquinone
O2
NAD(P)+
OH

O
O2

O
H3C
Reinicke et al., Clin. Cancer Res., 2005
CH3
Semiquinone
CH3
O2
70
2 min
10 min
60
50
40
30
20
10
0
5.0
7.5
10.0
15.0
ß-Lap Conc. (µM)
20.0
moles NADH Oxidized / mole Menadione
moles NADH Oxidized / moles ß-Lap
ß-Lapachone Induces a Futile Cycle of
NQO1-Mediated NADH Oxidation
70
2 min
10 min
60
50
40
30
20
10
0
10
40
50
75
85 100 200
Menadione Conc. (µM)
Pink et. al., JBC, 2000
ß-Lap Cytotoxicity: “Noncaspase-mediated Cell Death”
NAD(P)H
ß-Lapachone
(ß-Lap)
O
NAD(P)+
NQO1
ß-Lap
O2 Semiquinone
ß-Lap
Hydroquinone
O
O2
CH 3
O2
CH 3
ß-Lapachone (ß-Lap)
ROS
DNA Damage
Ca2+
PARP1
Hyper-activation
(NAD+/ATP Loss)
Repair
BAPTA-AM
Ca2+
(Chelation)
Ca2+
Influx
O2
O
AIF/µ-Calpain Activation
Endonuclease
Activation?
(CAD,Endo G, Acinus)
Nuclear
Condensation?
Caspase-Independent Apoptosis
NQO1-dependent ROS formation
Bentle et al., JBC, 2006; Cancer Res., 2007
Calcium Release After ß-Lap
ß-lap
0
1.5
3
4.5
3
6
12
7.5
13.5
9
10.5
Fold Increase
2.5
2
1.5
1
β-Lap
0.5
0
255
0
0
10
20
30
40
50
60
Time (min)
ß-lap
0
20 µM BAPTA-AM or 40 µM dicoumarol
1.5
3
4.5
3
6
12
7.5
13.5
9
10.5
Fold Increase
2.5
2
1.5
1
0.5
0
255
β-Lap
0
0
10
20
30
40
50
Time (min)
Tagliarino et al. JBC 276:19150, 2001
0
6
0
6
1.5
3
3
7.5 7.5
9
4.5
9 10.5
3
Fold Increase
TG
Calcium Release After ß-Lap is from ER stores
2.5
2
1.5
1
0.5
12
12 13.5 13.5
0
TG
ß-Lap
0
255
0
10
20
30
40
50
60
70
Time (min)
ß-lap
6
12
1.5
3
7.5
13.5
9
0
4.5
10.5
255
3
Fold Increase
0
2
1
ß-Lap
TG
0
0
10
20
30
40
50
60
70
80
Time (min)
Tagliarino et al. JBC 276:19150, 2001
NQO1-dependent, ß-Lap-induced DNA damage
γ-H2AX
Comet Assays
Bentle et al., JBC, 2006
ß-Lap Cytotoxicity: “Noncaspase-mediated Cell Death”
NAD(P)H
ß-Lapachone
(ß-Lap)
O2
NAD(P)+
NQO1
ß-Lap
O2 Semiquinone
ß-Lap
Hydroquinone
O2
O2
ROS
DNA Damage
Ca2+
PARP1
Hyper-activation
(NAD+/ATP Loss)
Repair
BAPTA-AM
Ca2+
(Chelation)
PARP in Action
β NAD+
Nuclear
protein
acceptor
Damaging
agents
PARP
Histones
Lamin B
PARP
Targets
Topo I,II
RPA
T-ag
DNA pol α
DNA pol β
XRCC-1
DNA Ligase IV
ATP
3-AB
β NAD+
Nicotinamide
+ H+
Nuclear
protein
acceptor
Rib Adenine
ADP-ribose
Adapted from http://parplink.u-strasbg.fr/index2.html
The Two Facets of PARP-1 Activation
Exogenous DNA
damaging
agents: γ, X IR
Alkylating agents
Limited DNA
damage
(replicating cells)
DNA
REPAIR
PARP-1
activation
DNA breaks
3-AB
Endogenous
free radicals
NO; ONOO
Saturated
DNA damage
(post-mitotic
cells,
Low NAD+
content)
PARP-1
over
activation
NECROPTOSIS
Nuclear condensation
DNA fragmentation
TUNEL +
Adapted from Shall S. and de Murcia G. (2000) Mut. Res. 460, 1 - 15
NAD+ depletion
ATP consumption
NQO1-dependent, PARP1 hyperactivation & nucleotide loss
NQO1+
MCF-7 Cells
MDA-MB-231 NQO1+
Bentle et al., JBC, 2006
PARP hyperactivation is necessary for ß-lap-induced apoptosis
1
Bentle et al.,
JBC, 2006
1 0.3 0.4
ß-Lap-induces DNA single strand breaks (SSBs)
Alkaline Comet Assay (Measures Total Breaks)
60 min
120 min
Neutral Comet Assay (Measures DSBs)
60 min
30 min
DMSO (120 min)
120 min
DMSO (120 min)
30 min
(4uM ß-lap treatment)
Ca2+ chelation allows repair and recovery after ß-lap
Bentle et al., JBC, 2006
ß-Lap Cytotoxicity: “Noncaspase-mediated Cell Death”
NAD(P)H
ß-Lapachone
(ß-Lap)
NQO1
ß-Lap
O2 Semiquinone
ß-Lap
Hydroquinone
O2
O2
ROS
DNA Damage
Ca2+
PARP1
Hyper-activation
(NAD+/ATP Loss)
Repair
BAPTA-AM
Ca2+
(Chelation)
Ca2+
Influx
O2
NAD(P)+
AIF/µ-Calpain Activation
Endonuclease
Activation?
Nuclear
Condensation?
Caspase-Independent Apoptosis
NQO1-dependent µ-calpain activation
NQO1NQO1-
p53 and atypical PARP1 cleavage
were hallmarks of ß-lap cell death
NQO1-
Proteolysis observed
12-24 h post-treatment
% Apoptosis
(%TUNEL T/C)
NQO1+
µ-Calpain Translocation to the Nucleus
DMSO
4h
6h
8h
+ DC
MCF-7 cells
DMSO
6h
8h
10 h
+ DC
MDA-468
-NQ3 cells
Anti-NQO1/PI
Control
MCF-7 (8 h)
β-Lap
MDA-468-NQ 3 (10 h)
Control
β-Lap
Tagliarino et al., Cancer Biol Ther., 2003
NQO1-dependent, Ca2+-regulated apoptosis inducing factor (AIF) activation
Bey et al., Unpub Data
Ca2+ chelation by BAPTA-AM pre-loading blocks AIF activation
Bey et al., Unpub Data
ß-Lap Cytotoxicity: “Noncaspase-mediated Cell Death”
NAD(P)H
ß-Lapachone
(ß-Lap)
NQO1
ß-Lap
O2 Semiquinone
ß-Lap
Hydroquinone
O2
O2
ROS
DNA Damage
Ca2+
PARP1
Hyper-activation
(NAD+/ATP Loss)
Repair
BAPTA-AM
Ca2+
(Chelation)
Ca2+
Influx
O2
NAD(P)+
AIF/µ-Calpain Activation
Endonuclease
Activation?
Nuclear
Condensation?
Caspase-Independent Apoptosis
Maximum antitumor therapeutic window for treating NSCLC with ß-lap
1.2
2h
0.8
1.2
4h
Relative Growth
T/C
Relative Growth
T/C
1.2
0.8
0.4
NQO1NQO1+
0.4
0.4
0
0
0
0
10
20
30
8h
0.8
40
0
0
10
ß-lap [µM]
20
30
10
40
20
30
40
§-lap 8h, [µM]
ß-lap [µM]
NQO1NQO1+
1.2
1.2
1.2
12 h
0.8
16 h
0.8
NQO1NQO1+
0.4
NQO1NQO1+
0.4
0
10
20
30
40
§-lap 12h, [µM]
NQO1NQO1+
0.4
0
0
24 h
0.8
0
0
10
20
30
§-lap 16h, [µM]
40
0
10
20
30
40
§-lap 24h, [µM]
H596 NSCLC cells
Bey et al., PNAS, 2007
Short pulses of ß-lap may increase its therapeutic index in treating
NSCLC
1.2
1.2
1.2
0.8
0.8
0.8
Dic
no Dic
Dic
no Dic
0.4
Dic
no Dic
0.4
0
0.4
0
0
5
10
15
20
0
0
§-Lap 2h, [µM]
5
10
15
20
0
5
10
15
20
§-Lap 8h, [µM]
§-Lap 4h, [µM]
1.2
1.2
0.8
0.8
Dic
no Dic
Dic
no Dic
0.4
0.4
0
0
0
5
10
15
§-Lap 16h, [µM]
20
0
5
10
15
20
§-Lap 24h, [µM]
A549 NSCLC cells
Bey et al., PNAS, 2007
β-Lapachone Redox Cycling
O
NAD(P)H
NAD(P)+
OH
Dicumarol
O
OH
NQO1
O
O
NAD(P)+
H3C
NAD(P)H
CH3
β-Lapachone

O2
H3C
P450R
b5R NAD(P)H
Hydroquinone
O2
NAD(P)+
OH

O
O2

O
H3C
Reinicke et al., Clin. Cancer Res., 2005
CH3
Semiquinone
CH3
O2
ß-Lapachone delivery methodology development for specific cancer therapies
I.
ß-Cyclodextrin
[Systemic administration]
II.
Millirods
[Brachytherapy, prostate cancer]
III. Nanoparticles (cRGD micelles)
[Lung Cancer, antiantiogenesis]
-Use Lung cancer-specific ligands
(e.g., αvß6) (Brown)
Cumulative Release
(ug/ml)
Polymer Microspheres
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
100
Time (Hours)
Scanning electron
micrograph
Release of Trypan blue
over 4 days
HP-ß-CD greatly improves ß-lapachone antitumor efficacy
ß-Lapachone antitumor responses using A549 cells
improve using an orthotopic model
Day 0
A549-Luc orthotopic model survival
% Animals Surviving
100
N=4/group
75
50
HP§-CD
§-lap-HP§-CD 30 mg kg
25
0
0
28
56
84
112 140 168 196 224 252
Time (Days)
End of
Therapy
ß-Lapachone is a potent radiosensitizer
HPßCD (Vehicle alone)
100
10 Gy
Mean Tumor Volume
(mm3)
600
ß-Lap (30 mg/kg)
10 Gy + ß-Lap
% Survival
75
400
200
50
25
*
* *
0
0
*p<0.0010
HP§CD
10 Gy
§-Lap-30 mg/kg
10 Gy+§-Lap
7
14
21
Time (Days)
A549 s.c. xenografts
0
7
14 21 28 35 42 49
Time (Days)
Dong et al. Fig. 4
A
B
1
3µM β-Lap+1Gy, min
1Gy, min
3µM β-Lap, min
LNCaP NQ+
UT 5 10 15 20 40 60 5 10 15 20
H2O2
40
60 V 5 10 15 20 40 60
PAR
α-tubulin
0.1
Gy
IR only
0 µM
1 µM + IR
1 µM2 µM + IR
2 µM3 µM + IR
Dq
1.25
0.95
0.7
0.38
D1
2
1.49
1.3
0.92
D0
0.88
0.68
0.62
0.6
3µM β-Lap+1Gy, min
1Gy, min
LNCaP NQ-
UT 5 10 15 20 40 60 5 10 15 20
3µM β-Lap, min
H2O2
40
60 V 5 10 15 20 40 60
3 µM
0.01
PAR
0
1
2
3
α-tubulin
IR (Gy)
C
a
120
100
b
120
100
80
80
60
60
40
40
2Gy
3µM
2Gy+3µM
20
0
0
DPQ
2Gy+3µM
2Gy+3µM+DPQ
20
0
60
120
0
60
§-Lap exposure (min)
§-Lap exposure (min)
120
c
120
100
80
60
60
40
40
3Gy
3µM
3Gy+3µM
20
d
100
80
DPQ
3Gy+3µM
3Gy+3µM+DPQ
20
0
120
0
0
60
§-Lap exposure (min)
120
0
60
§-Lap exposure (min)
120
Eva Cataldo
Bhavani Shankar, Ph.D.
Julio Morales, Ph.D.
Yonglong Zou, Ph.D.
Collaborators:
-Lindsey Mayo, Ph.D.
(Case)
-David Danielpour, Ph.D. (Case)
-David Chen, Ph.D.
(UTSW)
-Jerry Shay, Ph.D.
(UTSW)
-Sandeep Burma, Ph.D. (UTSW)
Tomoyuki Mashimo, Ph.D.
-H Evans, Ph.D.
(Case)
-M Watanabe, Ph.D. (Kiyoto)
-B Morgan, Ph.D.
(U. Maryland)
-D. Wilson, Ph.D.
(Case)
-C-R Yang, Ph.D.
(Case)
Acknowledgements
Collaborators
At UTSW:
J. Gao, PhD
J. Minna, Ph.D.
M. Peyton, Ph.D.
W. Bornmann, PhD
C. Thompson, MD, PhD
C. Distelhorst, MD
G. Dubyak, PhD
K. Brown, PhD
S. Ingalls
ERIK A. BEY
MELISSA BENTLE
KATE REINICKE