modulation of sphingolipid metabolism enhances apoptin’s cytotoxicity in prostate cancer
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Joseph C. ChengLaboratory of Dr. James S. Norris
Department of Microbiology & ImmunologyMedical University of South Carolina
October 31, 2007
Modulation of Sphingolipid Metabolism Enhances Apoptin’s Cytotoxicity in Prostate
Cancer
VP1 VP3VP2
Chicken Anemia Virus (CAV):
Apoptosis induction
VP1 No
VP2 Weak
VP3 Strong ApoptinApoptin
Noteborn, MH et al. (1994) JVI 68:346-351.
Nur77Nur77
ApoptinApoptin
How Apoptin Works in Cells
Crm1Crm1
ShieldingAggregationUbquitinationDegradation
PP
ApoptinApoptin
Nur77Nur77
ApoptinApoptin
PP
MitochondriaBcl-2Bcl-2
Nur77Nur77
Nur77Nur77
Nur77Nur77
Nur77Nur77
Nur77Nur77 Cytochrome C
Caspase 9
Caspase 3
Apoptosis
DEDAF
APC-1
Hippi
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
5' 3'
E1
E3 E4
SV40poly A
tTA
ITR
+ - tetracyclineor doxycycline
TETR +VP16
GFPApoptin TRE ITR
VP16
rTETR
SV40poly A
VP16
rTETR
VP16
rTETR
CMVPromoter
PC
-3
DU
145
LN
CaP
Bcl-2
Bax
FLIPL
FLIPS
DU
145
PC
-3
LN
CaP
Survivin
cIAP-1
XIAP
Bcl-xL
tubulin
Endogenous Gene Expression in Prostate Cancer Cells
DU145 (p53mt/mt), LNCaP (p53wt/wt), and PC-3 (p53null)
Liu et al. (2006) Mol Ther. 14:637-46.
Caspase 3
32KD
17KD
12KD
Ad-GFP Ad-Apop Ad-GFP Ad-Apop Ad-GFP Ad-Apop
DU145 LNcap PC3
Bak
Bax
P-p53
Actin
Apoptin Causes Caspase 3 Dependent Apoptosis in Prostate Cancer Cells
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.
CeramideCeramidases
Sphingosine
SphingosineKinase
S1P
Growth inhibition (cell cycle arrest)Apoptosis
Differentiation
Modulation of telomerase activity (telomere length)Senescence
(Pro-apoptotic phenotype)(Pro-apoptotic phenotype)
Cell proliferation
TransformationAngiogenesis
Cell motility (endothelial)
(Anti-apoptotic phenotype)(Anti-apoptotic phenotype)
S1PP
Stress (growth factor withdrawal, hypoxia,
hyperthermia, DNA damage)
Apoptin
Radiation
FasL/AdGFPFasLChemotherapy
0
20
40
60
80
100
120
140
160
180
200
0 10 20 30 40 50 60
Hours post-infection
% C
on
tro
lCeramide
Sphingomyelin
Sphingosine
Apoptin Causes Sphingolipids Changes in DU145 Cells
Liu et al. (2006) Mol Ther. 14:627-36.
Sphingomyelin De novo Synthesis
Ceramide
SMase
Ceramidases
Sphingosine
SphingosineKinase
S1P
Ceramide Synthase
S1PP
Apoptin
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)
Rig/S15
ASMase
Control 6hrs 16hrs 30hrs 48hrs Control 6hrs 16hrs 30hrs 48hrs
Ad-GFP Ad-Apoptin
AC
Western blot
ASMase
Ad-GFP Ad-GFPApoptin
Acid Ceramidase
Actin
Sphingomyelin
Ceramide
Sphingosine
ASMase
Acid Ceramidase
30 hours post-infection
Liu et al. (2006) Mol Ther. 14:627-36.
Translocation of Acid SMase by Confocal Microscopy Detection
16 hours post-infection
Ad-GFP
GFP (Green) ASMase (Red) Overlay
Ad-GFPApoptin
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)
20 30 40 50 60
MOI of Ad-Apoptin
Cel
l V
iab
ilit
y (%
)
0
10
20
30
40
50
60
70
80
90
* p<0.01
* p<0.01
Ad-GFPApoptinApoptin+ Desipramine (1 uM)Apoptin+Desipramine (2.5 uM)
Liu et al. (2006) Mol Ther. 14:627-36.
PKC delta-siRNAScrambled sequence siRNA
Ad-GFP
Ad-GFPApoptin
Ceramide level in PC3 Cells treated with PKC-siRNA
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
C-18:1-Cer C14-Cer C16-Cer C18-Cer C20-Cer C24-Cer C24:1-Cer
XL-1-Scramble
XL-1-pkc-SiRNA
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 pro- and 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.
Ceramide
Acid Ceramidase
Sphingosine Sphingosine-1-P
Sphingosine Kinase
Angiogenesis Anti-apoptosis
Ceramide/S1P Pathway
Pro- apoptosis
>60% Gleason grades 5-6 tissues over-express AC.>80% Gleason grades 8-10 tissues over-express AC.
Apoptin
Over-expression of AC Protect Apoptin’s Killing
Cytotoxicity of Ad-Apoptin in DU145 cells Over-expressing Acid Ceramidase
Cel
l Via
bili
ty
0
20
40
60
80
20 40 60 80 100 150
MOI
DU145-EGFP
DU145-ACEGFP#3
DU145-ACEGFP#7
Mock #3 #7
Liu et al. (2006) Mol Ther. 14:637-46.
HN
OH
HO NO2
. HCl
LCL204: Acid Ceramidase Inhibitor
LIPIDOMICS CORE
• 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.
Previous Studies:
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)
0
20
40
60
80
100
120
NT LCL 204 Ad-GFPApoptin Ad-GFPApoptin+LCL
Treatments
Cel
l Via
bil
ity
(%)
* #
* # ^
Liu et al. (2006) Mol Ther. 14:637-46.
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).
Design of in vivo experiments
0
100
200
300
400
500
600
700
800
0 5 10 15 20 25 30
Days
Rel
ativ
e tu
mor
vol
um
e
(% o
f or
igin
al)
Control
LCL-204
Apoptin
Apoptin+LCL
*#
*
Animal Study
Liu et al. (2006) Mol Ther. 14:637-46.
Days0 20 40 60 80
Su
rviv
al R
ate
(%)
0
20
40
60
80
100
LCL204
Apoptin
Control
Apoptin+LCL
Animal Study
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.
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