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Modulation of Sphingolipid Metabolism
Enhances Apoptin’s Cytotoxicity in Prostate
Cancer
Joseph C. Cheng
Laboratory of Dr. James S. Norris
Department of Microbiology & Immunology
Medical University of South Carolina
October 31, 2007
Chicken Anemia Virus (CAV):
VP1
VP2
VP3
Apoptosis induction
VP1
No
VP2
Weak
VP3
Strong
Apoptin
Noteborn, MH et al. (1994) JVI 68:346-351.
How Apoptin Works in Cells
Crm1
Shielding
Aggregation
Ubquitination
Degradation
Apoptin
Apoptin
Bcl-2
Mitochondria
Nur77
Nur77
Nur77
Nur77
Caspase 9
P
Apoptin
Cytochrome C
DEDAF
APC-1
Hippi
Caspase 3
Apoptosis
Prostate Cancer
• 217,000 new cases per year in U.S.,
(670,000 worldwide)
• 27,000 deaths per year in U.S.
• 2nd leading cause of cancer
death in American men.
• Improved methodologies of
diagnosis and treatment have
led to higher cure rate.
• Cancer-related deaths are due to advanced
disease by aggressive and resistant cancers.
AdGFPApoptinTET Vector
E1
ITR

E3 E4
tTA
CMV
Promoter
TETR + SV40
VP16 poly A
SV40
GFPApoptin
poly A
5'
TRE
VP16
VP16
rTETR
rTETR
+ - tetracycline
or doxycycline
VP16
rTETR
ITR
3'
FLIP
L
FLIP
S
PC-3
LNCaP
DU145
PC-3
LNCaP
DU145
Endogenous Gene Expression in Prostate Cancer Cells
Survivin
cIAP-1
XIAP
Bcl-2
Bcl-xL
Bax
 tubulin
DU145 (p53mt/mt), LNCaP (p53wt/wt), and PC-3 (p53null)
Liu et al. (2006) Mol Ther. 14:637-46.
Apoptin Causes Caspase 3 Dependent Apoptosis in Prostate Cancer Cells
DU145
Ad-GFP Ad-Apop
LNcap
PC3
Ad-GFP Ad-Apop Ad-GFP Ad-Apop
32KD
Caspase 3
17KD
12KD
Bak
Bax
P-p53
Actin
Liu et al. (2006) Mol Ther. 14:637-46.
Prostate Cancer Cell Lines Show Similar
Sensitivity to Ad-Apoptin
Liu et al. (2006) Mol Ther. 14:637-46.
Radiation
Stress (growth factor withdrawal, hypoxia,
hyperthermia, DNA damage)
Chemotherapy
FasL/AdGFPFasL
Apoptin
Ceramidases
Ceramide
Sphingosine
Sphingosine
Kinase
S1P
S1PP
(Pro-apoptotic phenotype)
Growth inhibition (cell cycle arrest)
Apoptosis
Differentiation
Modulation of telomerase activity (telomere length)
Senescence
(Anti-apoptotic phenotype)
Cell proliferation
Transformation
Angiogenesis
Cell motility (endothelial)
Apoptin Causes Sphingolipids Changes in DU145 Cells
Ceramide
200
Sphingomyelin
180
Sphingosine
160
% Control
140
120
100
80
60
40
20
0
0
10
20
30
40
50
60
Hours post-infection
Liu et al. (2006) Mol Ther. 14:627-36.
Apoptin
De novo Synthesis
Sphingomyelin
SMase
Ceramide Synthase
Ceramide
Ceramidases
Sphingosine
Sphingosine
Kinase
S1P
S1PP
The importance of sphingomyelin
hydrolysis in apoptosis
• Lymphoblasts derived from patients with acid SMase
deficiency (NPD), failed to undergo apoptosis in
response to irradiation or CD95 ligation.
• Radiation exposure of thymocytes from acid SMase
knockout mice did not undergo apoptosis.
• Ceramide generation induced by addition of
exogenous acid SMase augmented apoptosis in
human leukemic and prostate cancer cells.
Santana, P. et al. (1996) Cell 86:189.
De Maria, R. et al. (1998) J. Exp. Med. 187:897.
Monney, L. et al. Eur. J. Biochem. 251:295.
Condorelli, F. et al. (1999) Br. J. Pharmacol. 127:75.
RTPCR for Acid Sphingomyelinase (ASMase)/Acid Ceramidase (AC)
Ad-GFP
Control
ASMase
AC
Rig/S15
6hrs
16hrs
30hrs
Ad-Apoptin
48hrs
Control
6hrs
16hrs
30hrs
48hrs
Western blot
Ad-GFP
Ad-GFPApoptin
ASMase
Sphingomyelin
ASMase
Acid
Ceramidase
Ceramide
Acid
Ceramidase
Actin
Sphingosine
30 hours post-infection
Liu et al. (2006) Mol Ther. 14:627-36.
Translocation of Acid SMase by Confocal Microscopy Detection
GFP (Green)
ASMase (Red)
Overlay
Ad-GFP
Ad-GFPApoptin
16 hours post-infection
Liu et al. (2006) Mol Ther. 14:627-36.
Ad-Apoptin Increases ASMase Activity
MOI
Liu et al. (2006) Mol Ther. 14:627-36.
Desipramine Partly Delays Apoptin-induced Cell Death
DU145 Co-treated with Ad-Apoptin and Desipramine
(1uM and 2.5 uM)
90
Ad-GFPApoptin
Apoptin+ Desipramine (1 uM)
Apoptin+Desipramine (2.5 uM)
80
Cell Viability (%)
70
60
50
* p<0.01
40
* p<0.01
30
20
10
0
20
40
30
50
MOI of Ad-Apoptin
60
Liu et al. (2006) Mol Ther. 14:627-36.
Scrambled
sequence siRNA
Ad-GFPApoptin
Ad-GFP
PKC delta-siRNA
Ceramide level in PC3 Cells treated with PKC-siRNA
XL-1-Scramble
1.40
XL-1-pkc-SiRNA
1.20
1.00
0.80
0.60
0.40
0.20
0.00
C- 18:1- Cer
C14- Cer
C16- Cer
C18- Cer
C20- Cer
C24- Cer
C24:1- Cer
Summary
• Tumor-selective viral protein Apoptin induces
apoptosis in prostate cancer cells.
• There was no obvious correlation between
Apoptin-induced cell death and the status of proand anti-apoptotic molecules.
• Apoptin-mediated cell death involves modulation
of the sphingomyelin-ceramide pathway.
• Apoptin induces acid sphingomyelinase
translocation and activation through PKC.
• Inhibition of acid sphingomyelinase reduces the
efficacy of apoptin-induced cell death.
Apoptin
Ceramide/S1P Pathway
Acid
Ceramidase
Ceramide
Pro- apoptosis
Sphingosine Kinase
Sphingosine
Sphingosine-1-P
Angiogenesis
Anti-apoptosis
>60% Gleason grades 5-6 tissues over-express AC.
>80% Gleason grades 8-10 tissues over-express AC.
Over-expression of AC Protect Apoptin’s Killing
Cytotoxicity of Ad-Apoptin in DU145 cells
Over-expressing Acid Ceramidase
80
Mock #3
#7
Cell Viability
60
40
DU145-EGFP
20
DU145-ACEGFP#3
DU145-ACEGFP#7
0
20
40
60
80
100
150
MOI
Liu et al. (2006) Mol Ther. 14:637-46.
LCL204: Acid Ceramidase Inhibitor
OH
HO
NO
HN
2
.
H Cl
LIPIDOMICS CORE
Previous Studies:
• AC inhibition by LCL204 results in ceramide
accumulation and conversion from an anti-apoptotic
phenotype to a pro-apoptotic phenotype.
• LCL204 displays lysomotropic properties by
causing rapid lysosomal membane permeabilization
(LMP) resulting in translocation of the lysosomal
proteases cathepsins B and D into the cytosol.
• Apoptosis induced by LCL204 is dependent on
Bak, suggesting that LMP induces a mitochondrial
apoptotic pathway.
• LCL204 significantly down-regulates anti-apoptotic
genes Flip and Survivin.
Holman et al. 2007 Cancer Chemother Pharmacol DOI: 10.1007/s00280-007-0465-0
Acid Ceramidase Inhibitor LCL204 Enhanced Apoptin’s Effect
DU145 Cells Treated with Ad-GFPApoptin (MOI 20))
and Followed by LCL204 (5 uM)
120
Cell Viability (%)
100
80
*#
60
40
*#^
20
0
NT
LCL 204
Ad-GFPApoptin
Ad-GFPApoptin+LCL
Treatments
Liu et al. (2006) Mol Ther. 14:637-46.
Design of in vivo experiments
Tumors are treated with 5 intraperitoneal injections of
LCL204 75 mg/kg (Q 3 days).
Tumors are treated with 4 intratumoral injections of
2 X 109 PFU adenovirus (Q 3 days).
Animal Study
800
Control
700
LCL-204
Apoptin
600
(% of original)
Relative tumor volume
Apoptin+LCL
500
*
400
300
*
#
200
100
0
0
5
10
15
Days
20
25
30
Liu et al. (2006) Mol Ther. 14:637-46.
Animal Study
100
Survival Rate (%)
80
60
40
Control
LCL204
20
Apoptin
Apoptin+LCL
0
0
20
40
60
80
Days
Liu et al. (2006) Mol Ther. 14:637-46.
Summary
• Apoptin-mediated cell death involves modulation
of the sphingomyelin-ceramide pathway.
• Ceramide accumulates in response to Apoptin via
increased biosynthesis (ASMase) and retention
(AC).
• Pretreatment of prostate cancer cells with AC
inhibitor sensitizes tumors to Apoptin, indicating AC
is a potential therapeutic target.