Impact of DNA Repair Mechanisms on Resistance to alkylating therapeutic agents
TMZ
MTIC
N
N N
C
N
N
O N
NH2
CH3
O
N
C
N
O N
NH2
CH3
N
CH3
N
C
N
O N
NH2
H
N
CH3
DITC
H2N
O CH3
N
N
N
N
O6mG
N7mG
N
C
N
O N
NH2
H
N
CH3
H2N
O CH3
N
N
N
NMTIC
TMZ
Repair Pathways Responsible for ProcessingMethyl DNA Adducts
TMZ
O6mG (6%)
N7mG (70%)N3mA (9%)
MGMT & MMR
BER
Direct Repair of O6mG Mediated by AGT
AGT
0.1 % 1 % 10 % 100 %
0
500
1000
1500
0 10 20 30 40 50 60Days
Tu
mor
Volu
me (
mm
3)
100 10 1 0.1 0
The Correlation of MGMT Activity to the Drug Resistance
BCNU 30 mg/kg
Cancer Res. 1997
H2N
O
N
N
N
HN
Reaction of O6-benzylguanine and AGT Protein
MGMT
CysS
MGMT
CysS
H2N
O
N
N
N
N
-
O6-benzylguanine (BG) Guanine
O6-Methylating Agent
AGT
Mismatch Repair
Cytotoxicity Cell Survival
AGT
MGMT Repair
O6mGT/C
O6mG
MSH6
O6mG
Cytotoxicity
Mismatch Repair
T/C
MSH2
MLH1
PMS2
MSH2
MutH
1
10
100
0 500 1000 1500 20001
10
100
0 500 1000 1500 2000
TMZ(µM)
1
10
100
0 500 1000 1500 2000
SW480 (MMR+) HCT 15 (MSH6 mut) HCT 116 (MLH1 mut)
Resistance of MMR Deficient Colon Cancer Cells to TMZ
+BG
+BG
+BG
Cancer Res. 1996
Repair Pathways Responsible for ProcessingMethyl DNA Adducts
TMZ
O6mG (6%)
N7mG (70%)N3mA (9%)
MGMT & MMR
BER
AP site
MPG
APE
BER pathway N7mG
XRCC1
XRCC1
XRCC1
PARP
DNA pol
LigaseIII
Methoxyamine
MX
CO H
APE
5’ P P P 3’
MPG
5’ P P P 3’
CH3
CHO H
HNO
5’ P P P 3’
Methoxyamine binds to aldehyde group in an AP site
AP site
AP-MX site AP SiteAP-MX Site
APE+ - + -
MX bound AP Site is Resistant to Cleavage by AP-endonuclease
UDG
MX
NHOCH3
U
40 mer
20 mer
AP site-ARPAP site3’
OH5’
O
3’
OH5’
N O StrepHRP
NN
O
H
H
OO
NH2
Strepta.HRP
0
1
2
3
4
5
6
0 0.375 0.75 1.5 3 6
r2 = 0.993Un
it o
f D
ensi
ty
AP-DNA substrate (g)
3’
OH5’
N O
AP site-MX3’
OH5’
O
AP site
NH-O- CH3 CH3
AP
sit
es
(un
it o
f d
ensi
ty)
TMZ (uM)TMZ (uM)
0
2
4
6
8
10
12
14
0 187 375 750 1500
TMZ
TMZ+ MX
0
2
4
6
8
10
12
14
0 187 375 750 1500
TMZ
TMZ+ MX
MX-AP
Control
TMZ (175µM)
TMZ (175µM)+MX(25 mM)
TMZ (350µM)
TMZ (750µM)
TMZ (350µM)+MX(25 mM)
TMZ (750µM)+MX(25 mM) 0
2.5
5
7.5
10
0 94 187 375 750 1500
r = 0.995
TMZ(µM)
Cell Death
Block DNA Synthesis
Methoxyamine Interrupt BER Pathway
CH3
P P P 3’5’
CHO H
HNO
ChromosomalAberration
Apoptosis
MX APE
0
500
1000
1500
2000
0 25 50 75
Days
MX+TMZ
BG (30mg/kg)+TMZ
TMZ(120mg/kg)
MX (0.2mg/kg)
Control
SW480 Tumor Growth in Nude Mice
Clin. Cancer Res. 2002
0
500
1000
1500
2000
0 25 50 75 100
Days
BG+MX+TMZ
MX (0.2 mg/kg)+TMZ
BG (30 mg/kg +TMZ)
TMZ 40mg/kg
Control
Inhibition of SW480 Tumor GrowthBy BG plus MX and TMZ in Nude Mice
HCT116 Tumor Growth in Nude Mice
0
500
1000
1500
2000
0 5 10 15 20 25 30
Days
MX+TMZ
MX (0.2 mg/kg)
BG (30 mg/kg)+TMZ
TMZ (120 mg/kg)
Control
Compared to TMZ alone, the combination of MX+TMZ has No additive toxicity,
Compared to BG+TMZ, the combination of MX+TMZ has No toxic death
Less body weight loss
Less myelosuppression
Blocking BER efficiently potentiate TMZ-antitumorEffect bypassing MMR and p53 genetic status.
Liu & Gerson. Clin Cancer Res 2006;12:328-331
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