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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 ¨C 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
0X 250X 100X 50X
0
25
50
75
100
CytoReg
CytoReg (pH 7.4)
Dilution
Cellviability(%ofcontrol)
Cytoreg vs. Cyto
0X 1000X 500X 250X 100X 50X
0
25
50
75
100 CytoReg
CytoRegM
Dilution
Cellviability(%ofcontrol)
FIGURE 2
(A) Flow cytometry in T98G/U87MG ¨C 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)
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)
FIGURE 4
(A) PI staining showing membrane disruption ¨C 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
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)
FIGURE 6
Effect of CytoReg on primary brain tumors from patients ¨C 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

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GBM Cancer Cells Reserach Report MCG 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 ¨C 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 ¨C 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 ¨C 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 ¨C 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