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Reactive oxygen species-mediated
intercellular signaling of malignant cells:
Control of multistep oncogenesis and a
chance for novel therapeutic approaches.
Georg Bauer University of Freiburg, Germany
Nontransformed cell
Transformed cell
Oncogene activation
Tumor suppressor gene inactivation
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
Extracellular reactive oxygen species drive proliferation and maintain the transformed state!
Superoxide anions
src ras rac
NADP+ + H+
2 e-
NADPH - O2 .
O2 O2
- O2 .
H2O2
Proliferation stimulus
2 H +
Procarcinogenic effects of ROS: A) initiation of transformation through mutagenesis, B) maintenance of the transformed state through extracellular superoxide anions Anticarcinogenic effects of ROS: Elimination of transformed cells through intercellular ROS signaling and apoptosis induction
Transformed target cells
Intercellular induction of apoptosis
Transformed target cells (high local density)
Intercellular induction of apoptosis Nontransformed effector cells
Transformed target cells (high local density)
Intercellular induction of apoptosis Nontransformed effector cells
Apoptosis
Transformed cells only Autocrine apoptosis induction
Target cell function (high local cell density required)
Autocrine apoptosis induction Effector cell function
Autocrine apoptosis induction
Apoptosis
Apoptotic cells
Nontransformed cell
Transformed cell
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
Apoptosis
Elucidation of intercellular ROS signaling: 1) Inhibitor studies
2) Reconstitution experiments
3)Verification of reconstitution experiments with inhibitors 4) siRNA-based analysis
Inhibitor studies
Hydroxyl radical scavenger
Peroxidase-Inhibitor HOCl-Scavenger
Superoxide anion scavenger
H2O2 + Cl- HOCl + OH-
HOCl + O2.- .OH + Cl- + O2
POD
HOCl signaling pathway
.NO + O2.- ONOO-
ONOO- + H+ ONOOH
ONOOH .NO2 + .OH
NO/peroxynitrite signaling pathway
Signaling pathways
Basic features
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
Intercellular space
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
Intercellular space
Autocrine ROS
signaling
Further elucidation
of ROS signaling pathways
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
ABH
AEBSF Apocynin
L-NAME 3-Br-7-NI
SOD MnTM-2PyP
FeTPPS
CAT EUK-134
Mannitol
Taurine
Inhibitor studies
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
NO
ONOO_ POD
Addition of signaling molecules (Reconstitution experiments)
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
X
X
X X X
X
X
X
X
X
SiRNA-mediated knockdown
HOCl signaling pathway
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
NO/peroxynitrite signaling pathway
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Apoptosis
Apoptosis
Transformed
target cell
Nontransformed
effector cell O2 . -
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
6
9
11
7
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
17
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Transformed
target cell
Arginase
MMP
RAS RAC Active TGF-beta-1
Inactive TGF-beta-1
TGF-beta-R
LPO
LPO
1 2
8
3
4 5
10
12
13
14
15
16
18 Details of autocrine
ROS signaling
Bauer, 2012
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
6
9
11
7
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
17
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Transformed
target cell
Arginase
MMP
RAS RAC Active TGF-beta-1
Inactive TGF-beta-1
TGF-beta-R
LPO
LPO
1 2
8
3
4 5
10
12
13
14
15
16
18
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
6
9
11
7
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
17
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Transformed
target cell
Arginase
MMP
RAS RAC Active TGF-beta-1
Inactive TGF-beta-1
TGF-beta-R
LPO
LPO
1 2
8
3
4 5
10
12
13
14
15
16
18
Intracellular pathways
activated by intercellular
ROS-mediated signaling
NO2 .
OH .
HOCl
- Cl - ONOO
- O2 .
- O2 .
- O2 . - O2 . - O2 .
H2O2
Arginine Citrulline
NO . H+
ONOOH
NO .
OH .
Apoptosis
LPO
LPO
SMase
Ceramide
PTT CytC
APAF
CytC
Procaspase-9
Caspase-9
Caspase-3 Procaspase-3
Intracellular signaling pathways after intercellular ROS-mediated signaling
extracellular
intracellular
Nontransformed cell
Transformed cell
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
autocrine
intercellular
Apoptosis
Nontransformed cell
Transformed cell
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
autocrine
intercellular
Apoptosis
Elimination of transformed cells: potential protective mechanism against tumor formation?
Requirements for tumor progression:
Maintaining proliferation stimulus of ROS
and
Protection against apoptosis induction
by ROS
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 .
Tumor cell
CAT CAT
CAT CAT
Resistance against intercellular ROS signaling
Mechanism of tumor cell resistance
Membrane-associated catalase
Nontransformed cell
Transformed cell
Data obtained in collaboration with Galina Deichman, Moscow
Role of membrane-associated catalase
for the protection of tumor cells
against ROS signaling.
How can catalase protect against
multiple signaling pathways?
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Arginase
Catalase protects tumor cells against HOCl and NO/peroxynitrite signaling
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
Heinzelmann and Bauer, 2010
Classical catalase activity
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
Heinzelmann and Bauer, 2010
Classical catalase activity
Additional catalase functions
H2O + O2
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2
Catalase is multifunctional!
Utilization of ROS signaling
chemistry for novel,
mechanism-based
antitumor strategies
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 . - O2 .
- O2 .
- O2 .
- O2 .
- O2 . - O2 .
- O2 .
Tumor cell
CAT CAT
CAT CAT
Inhibition or inactivation of CAT
siCAT Intercellular ROS signalling
Fibrosarcoma
Fibrosarcoma plus Catalase inhibitor
Ovarial carcinoma cell line
Ovarial carcinoma cell line after catalase inactivation
Distinct ways to sensitize
tumor cells for
apoptosis induction
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Arginase
INH
1. Inhibition of catalase by 3-aminotriazole
3-Aminotriazol
0
10
20
30
40
50
0
3,1
6,2
12,5 25 50 75
100
150
200
FeTPPS
L-NAME
Mn-SOD
ABH
AEBSF
MANNITOL
TAUCONTROL
AP
OP
TOTI
C C
ELL
S (
% )
3-AT (mM)
Scavenger for superoxide anions and inhibitor of NADPH oxidase
NO/peroxynitrite pathway HOCl pathway
0
10
20
30
40
0 0,64 1,6 4 9,6 24
siCAT (nM)
L-NAME
TAU
ABH
Mn-SOD
WITHOUT INH
AP
OP
TOTI
C C
ELL
S (
% )
siRNA-mediated knockdown of catalase sensitizes tumor cells for ROS signaling
2. SiRNA-mediated knockdown of catalase
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Arginase
1O2
PHOTOFRIN
x
Light
1O2
1O2
3. Singlet oxygen-mediated inactivation of catalase
Apoptosis
Increase in the cellular NO concentration (through addition of arginine, inhibition of arginase or inhibition of NO dioxygenase) causes generation of extracellular singlet oxygen of the tumor cells. Singlet oxygen inactivates catalase and thus restores apoptotic ROS signaling specifically in tumor cells.
This process requires amplification through APO/FAS receptor mediated stimulation of NOX1 and NOS.
4. Inactivation of catalase by tumor cell-derived singlet oxygen
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
. NO
. NO
. NO
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
FAS-R
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
FAS-R NOX-1
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
FAS-R NOX-1
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
FAS-R NOX-1
1O2
1O2
CAT Fe III CAT Fe IV = O+. CAT Fe III
H2O
- NO2
H2O2 H2O + O2
ONOO -
. NO . NO2
ONOO - - NO2 + O2
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2 1O2 1O2
1O2
CAT Fe III
ONOO -
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
x
1O2 1O2
1O2
CAT Fe III
ONOO -
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
x
1O2
1O2
1O2
1O2 1O2 1O2
CAT Fe III
ONOO -
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2 Cl -
O2 . - 2 + 2 H+
CAT Fe III NO
. NO
. NO
. NO
1O2
x
1O2
1O2
1O2
1O2 1O2 1O2
More singlet oxygen generation More catalase inactivation Restoration of intercellular apoptotic ROS signaling
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Arginase
5. Inhibition of catalase by mAB sensitizes tumor cells for ROS signaling
Apoptosis
mAb
HOCl
- Cl
H2O2 + O2
+ OH- +2 H+
.OH + Cl- + O2
NO .
- ONOO
Arginine Citrulline
ONOOH H+
.OH
NO2 .
H2O + O2 +H+ +Cl-
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
O2 . -
Arginase
In addition to restoration of ROS signaling, anti-CAT triggers dynamic, automplificatory inactivation of catalase by singlet oxygen (next page)
CAT
x ONOO -
H2O2
ONOO -
ONOO -
ONOO - H2O2
H2O2
H2O2
ONOO - H2O2
CAT
x 1O2
1O2 ONOO -
H2O2
ONOO -
ONOO -
ONOO - H2O2
H2O2
H2O2
CAT
x 1O2
1O2 ONOO -
H2O2
ONOO -
ONOO -
ONOO - H2O2
H2O2
H2O2
CAT
x ONOO -
H2O2
ONOO -
ONOO -
ONOO - H2O2
H2O2
H2O2
CAT
x ONOO -
H2O2
ONOO -
ONOO -
ONOO - H2O2
H2O2
H2O2
1O2 1O2
CAT
x ONOO -
H2O2
ONOO - ONOO - H2O2
H2O2
H2O2
1O2 1O2
CAT
x ONOO -
H2O2
ONOO - ONOO - H2O2
H2O2
H2O2
CAT
x
ONOO -
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2
Cl -
O2 . - 2 + 2 H+
CAT
x
ONOO -
H2O2+ O2
. NO
O2 . -
ONOOH
. NO2 + .OH
H+
POD
HOCl
O2 . -
. OH + Cl - + O2
Cl -
O2 . - 2 + 2 H+
Apoptosis
For potential future therapeutic applications we focus on i) recombinant neutralizing antibodies, ii) compounds that trigger extracellular singlet oxygen generation by tumor cells; iii) the utilization of synergy effects between these compounds. These approaches are highly specific for the ROS-relevant phenotype of tumor cells (NOX1 activation combined with expression of membrane-associated catalase) and do not affect intracellular catalase of nonmalignant cells.
All three approaches have been extensively evaluated in vitro. They have been proven valid for tumor cells derived from the most frequent as well as the most aggressive tumors. Our concepts for novel tumor therapy, based on restoration of intercellular ROS signaling, are protected by several patents and patent applications. First proof-of-concept experiments in vivo have been successful.
Summary: Transformed cells generate extracellular superoxide anions. Extracellular superoxide anions and their dismutation product H2O2 drive the proliferation of malignant cells. They are also the basis for the HOCl and the NO/peroxynitrite signaling pathway that selectively eliminate transformed cells. Tumor progression requires resistance against apoptosis-inducing ROS signaling.
Resistance of tumor cells is acquired through expression of membrane-associated catalase. Catalase inhibits HOCl signaling through decomposition of H2O2 and interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. Inhibition of membrane-associated catalase of tumor cells with monoclonal antibodies or its inactivation through singlet oxygen causes ROS-mediated apoptotic selfdestruction of the tumor cells. This efficient and highly selective principle is a promising approach for novel strategies for tumor therapy.
I am grateful to Manfred Saran Reinhold Schäfer Galina Deichman for sharing their concepts, ideas and biological systems.
Our work was funded by DFG Deutsche Krebshilfe Clotten-Stiftung RiscRad EuroTransBio Hans Sauer Stiftung
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