This Presentation shows the effect of Cytoreg(R) in various Brian Tumor Cell lines. Performed at the Medical College of Georgia. Presented in April of 2008

1. 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

2. 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
Control
1000X
500X
250X
100X 50X
25X
75X
Treatment Time
Cellviability(%ofcontrol)
0.5h 1h 3h 24h 48h
0
25
50
75
100
C ontrol
1000X
500X
250X
100X
50X
25X
75X
Treatment Time
Cellviability(%ofcontrol)
T98G

3. 0X 250X 100X 50X
0
25
50
75
100
CytoReg
CytoReg (pH 7.4)
Dilution
Cellviability(%ofcontrol)

4. Cytoreg vs. Cyto
0X 1000X 500X 250X 100X 50X
0
25
50
75
100 CytoReg
CytoRegM
Dilution
Cellviability(%ofcontrol)

5. 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
125
Vehicle
250X CytoReg
CytoReg + Z-VAD.fmk
CytoReg + PFT-
Cellviability(%ofcontrol)

6. FIGURE 3
Effect of CytoReg on non-transformed cells (neurons, astrocytes)
Neurons Astrocytes
0
25
50
75
100
125
Veh
50X
100X
250X
Cellviability
(%ofcontrol)

7. FIGURE 4
(A) PI staining showing membrane disruption – 6h T98G
(B) Membrane disruption assay
(C) Add trypan blue staining
0x 250X 100X
Data 1
0X 250X 100X
0
30
60
90
0X
250X
100X
CytoReg
%CellularMembrane
Disruption
0X 250X 100X
0
25
50
75
100
CytoReg
%TrypanBluePositive

8. FIGURE 5:
KM D-456b Effect of CytoReg on BCNU-induced cell death in
T 98G gliom a cells following a 72h treatm ent in serum
0 20 40 80
1
10
100
1000X CytoReg
500X CytoReg
0X CytoReg
BCNU ( M)
Cellviability(%ofcontrol)
KM D-456 Effect of CytoReg on BCNU-induced cell death
U87M G gliom a cells following a 72h treatm ent in serum
0 20 40 80
1
10
100
0X CytoREG
1000X CytoREG
500X CytoREG
BCNU ( M)
Cellviability(%ofcontrol)
KM D-448 Effect of CytoReg in com bination w ith T em odar
follow ing a 48h treatm ent in confluent T 98G glioblastom a cells
0 50 100 250
10
100
0X
1000X
500X
*
**
**
Temozolomide ( M)
Cellviability
(%ofcontrol)

9. 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
Cellularviability(%ofcontrol)
GBM #1
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Cellularviability(%ofcontrol)
GBM #2
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Cellularviability(%ofcontrol)
24h 48h
0
25
50
75
100 Control
1000X
500X
250X
100X
75X
50X
25X
Cellularviability(%ofcontrol)
Men #1
Men #2