reactive oxygen species -mediated intercellular … · reactive oxygen species -mediated...

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


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


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


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


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


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


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

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


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


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


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+


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


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


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


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


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


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


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 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

reactive oxygen species -mediated intercellular … · reactive oxygen species -mediated...

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