modulation of sphingolipid metabolism enhances apoptin’s cytotoxicity in prostate cancer
DESCRIPTION
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. Apoptin. Chicken Anemia Virus (CAV):. VP1. VP3. VP2. - PowerPoint PPT PresentationTRANSCRIPT
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.