gbm cancer cells reserach report mcg 2008
TRANSCRIPT
Cytoreg® induced cytotoxicity in
human brain tumors involves
acute membrane damage
Sangeetha Sukumari-Ramesh1, Nagendra Singh2, Suash Sharma2,
John R. Vender1, Krishnan M. Dhandapani1
1Department of Neurosurgery and 2 Department of Pathology,
Medical College of Georgia
FIGURE 1
(A) Effect of CytoReg (concentration response) on T98G, U87MG, T67, C6 glioma
cells by MTT (and LDH – data not shown)
(B) Cell Morphology
0.5h 1h 3h 6h 24h 48h
0
25
50
75
100
C ontrol
1000X
500X
250X
100X 50X
25X75X
Treatment Time
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
0.5h 1h 3h 24h 48h
0
25
50
75
100
C ontrol
1000X
500X
250X
100X50X
25X75X
Treatment Time
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
T98G
0X 250X 100X 50X
0
25
50
75
100Cy toReg
Cy toReg (pH 7.4)
Dilution
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
Cytoreg vs. Cyto
0X 1000X 500X 250X 100X 50X
0
25
50
75
100 C y toReg
C y toRegM
D ilution
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
FIGURE 2
(A) Flow cytometry in T98G/U87MG – necrosis vs. apoptosis
(B) Western of cleaved caspase3, DNA fragmentation
T98G U87MG
0
25
50
75
100
125Vehic le
250X Cy toReg
Cy toReg + Z-VAD.fmk
Cy toReg + PFT-
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
FIGURE 3
Effect of CytoReg on non-transformed cells (neurons, astrocytes)
Neurons Astrocytes
0
25
50
75
100
125Veh
50X
100X
250X
Ce
ll v
iab
ilit
y
(% o
f c
on
tro
l)
FIGURE 4
(A) PI staining showing membrane disruption – 6h T98G
(B) Membrane disruption assay
(C) Add trypan blue staining
0x 250X 100X
D ata 1
0X 250X 100X
0
30
60
900X
250X
100X
C ytoR eg
% C
ell
ula
r M
em
bra
ne
Dis
ru
pti
on
0X 250X 100X
0
25
50
75
100
CytoReg
% T
ry
pa
n B
lue
Po
sit
ive
FIGURE 5:
K M D -456b E ffect o f C ytoReg on B C NU-induced ce ll death in
T 98G g liom a ce lls fo llow ing a 72h treatm ent in serum
0 20 40 80
1
10
100
1000X Cy toReg
500X Cy toReg
0X Cy toReg
BCNU ( M)
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
K M D -456 E ffect o f C ytoReg on B C NU-induced ce ll death in
U87M G g liom a ce lls fo llow ing a 72h treatm ent in serum
0 20 40 80
1
10
100
0X Cy toREG
1000X Cy toREG
500X Cy toREG
BCNU ( M)
Ce
ll v
iab
ilit
y (
% o
f c
on
tro
l)
K M D -448 E ffect o f C ytoReg in com bination w ith T em odar
fo llow ing a 48h treatm ent in confluent T 98G g liob lastom a ce lls
0 50 100 250
10
100
0X
1000X
500X*
**
**
Temoz olomide ( M)
Ce
ll v
iab
ilit
y
(% o
f c
on
tro
l)
FIGURE 6
Effect of CytoReg on primary brain tumors from patients – 3 GBM and 5
meningiomas -
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Ce
llu
lar v
iab
ilit
y (
%o
f c
on
tro
l)
GBM #1
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Ce
llu
lar v
iab
ilit
y (
%o
f c
on
tro
l)
GBM #2
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Ce
llu
lar v
iab
ilit
y (
%o
f c
on
tro
l)
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Ce
llu
lar v
iab
ilit
y (
%o
f c
on
tro
l)
Men #1
Men #2