Georg Bauer University of Freiburg, Germany

Transcription

1 Reactive oxygen speciesmediated intercellular signaling of malignant cells: Control of multistep oncogenesis and a chance for novel therapeutic approaches Georg Bauer University of Freiburg, Germany

2 Nontransformed cell Transformed cell proliferation and maintain the transformed state! O Oncogene activation O Extracellular reactive oxygen species drive O O O O O Superoxide anions Tumor suppressor gene inactivation

3 Proliferation stimulus NADP + + H + H O src ras rac NADPH e O O H + O O

4 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

5 Intercellular induction of apoptosis Transformed target cells

6 Intercellular induction of apoptosis Nontransformed effector cells Transformed target cells (high local density)

7 Intercellular induction of apoptosis Nontransformed effector cells Transformed target cells (high local density) Apoptosis

8 Autocrine apoptosis induction Transformed cells only

9 Autocrine apoptosis induction Effector cell function Target cell function (high local cell density required)

10 Autocrine apoptosis induction Apoptosis

11 Apoptotic cells

12 Nontransformed cell Transformed cell O O O O O O O Apoptosis

13 Elucidation of intercellular ROS signaling: 1) Inhibitor studies ) Reconstitution experiments 3)Verification of reconstitution experiments with inhibitors 4) sirnabased analysis

14 Inhibitor studies

15 PeroxidaseInhibitor HOClScavenger Superoxide anion scavenger Hydroxyl radical scavenger

16 POD H O + Cl HOCl + OH HOCl + O OH + Cl + O HOCl signaling pathway

17 NO + O ONOO + H + ONOOH ONOO ONOOH NO + OH NO/peroxynitrite signaling pathway

18 Signaling pathways

19 Basic features

20 O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO Cl O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O Apoptosis O H O + O +H + +Cl Transformed target cell Intercellular space

21 O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO Cl O O H O + O + H + O HOCl + OH Autocrine ROS Arginine Citrulline OH + Cl + O signaling Apoptosis O H O + O +H + +Cl Transformed target cell Intercellular space

22 Further elucidation of ROS signaling pathways

23 Inhibitor studies O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO CAT EUK134 FeTPPS Cl ABH LNAME 3Br7NI AEBSF Apocynin O O H O + O + H + O Taurine HOCl + OH Mannitol Arginine Citrulline Apoptosis OH + Cl + O SOD H O + O +H + +Cl O MnTMPyP Transformed target cell

24 ONOO _ POD Addition of signaling molecules (Reconstitution experiments) NO O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO Cl O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O Transformed target cell Apoptosis O H O + O +H + +Cl

25 SiRNAmediated knockdown X Nontransformed O NO effector cell Apoptosis X ONOOH H + NO OH X O ONOO X O O H O + O + H + O Cl X HOCl + OH X X Arginine Citrulline OH + Cl + O Transformed target cell Apoptosis O X X X H O + O +H + +Cl

26 HOCl signaling pathway

27 O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO Cl O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O Apoptosis O H O + O +H + +Cl Transformed target cell

28 NO/peroxynitrite signaling pathway

29 O NO Nontransformed effector cell Apoptosis ONOOH H + NO OH O ONOO Cl O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O Apoptosis O H O + O +H + +Cl Transformed target cell

30 17 O 16 NO 18 Details of autocrine ROS signaling LPO ONOOH H + NO OH O ONOO 8 Cl 15 Arginase 13 Arginine 14 Citrulline 6 O O H O + O + H O 9 HOCl + OH 10 OH + Cl + O 1 RAS 1 RAC LPO O O 3 H O + O +H + +Cl Active TGFbeta1 Bauer, 01 Transformed target cell 5 MMP TGFbetaR 4 Inactive TGFbeta1

31 17 O 16 NO 18 LPO ONOOH H + NO OH O ONOO 8 Cl 15 Arginase 13 Arginine 14 Citrulline 6 O O H O + O + H O 9 HOCl + OH 10 OH + Cl + O 1 RAS 1 RAC LPO O O 3 H O + O +H + +Cl Active TGFbeta1 Transformed target cell 5 MMP TGFbetaR 4 Inactive TGFbeta1

32 17 O 16 NO 18 LPO ONOOH H + NO OH O ONOO 8 Cl 15 Arginase 13 Arginine 14 Citrulline 6 O O H O + O + H O 9 HOCl + OH 10 OH + Cl + O 1 RAS 1 RAC LPO O O 3 H O + O +H + +Cl Active TGFbeta1 Transformed target cell 5 MMP TGFbetaR 4 Inactive TGFbeta1

33 Intracellular pathways activated by intercellular ROSmediated signaling

34 Intracellular signaling pathways after intercellular ROSmediated signaling intracellular O LPO ONOO O NO H + NO OH NO ONOOH H O O O O Cl HOCl extracellular Arginine Citrulline SMase OH Ceramide LPO Apoptosis PTT CytC CytC Caspase3 Procaspase3 APAF Procaspase9 Caspase9

35 Nontransformed cell Transformed cell Apoptosis O O O O O O O autocrine intercellular

36 Nontransformed cell Transformed cell Apoptosis O O O O O O O autocrine intercellular Elimination of transformed cells: potential protective mechanism against tumor formation?

37 Requirements for tumor progression: Maintaining proliferation stimulus of ROS and Protection against apoptosis induction by ROS

38 Mechanism of tumor cell resistance Nontransformed cell Transformed cell Tumor cell Membraneassociated catalase O O O O O O O O CAT CAT O O CAT O O CAT O Resistance against intercellular ROS signaling Data obtained in collaboration with Galina Deichman, Moscow

39 Role of membraneassociated catalase for the protection of tumor cells against ROS signaling

40 How can catalase protect against multiple signaling pathways?

41 Catalase protects tumor cells against HOCl and NO/peroxynitrite signaling O NO ONOOH H + NO OH O ONOO Cl Arginase O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O O H O + O +H + +Cl

42 Cl POD O + H + O HOCl H O + O OH + Cl + O Classical catalase activity H O H O H O + O CAT Fe III CAT Fe IV = O + CAT Fe III NO ONOO H + NO ONOO NO + O O ONOOH NO + OH NO NO Heinzelmann and Bauer, 010

43 Cl O HOCl OH + Cl + O POD O + H + H O + O Classical catalase activity H O H O H O + O CAT Fe III CAT Fe IV = O + CAT Fe III NO ONOO H + NO ONOO NO + O O ONOOH NO + OH NO NO Additional catalase functions Heinzelmann and Bauer, 010

44 Catalase is multifunctional! H O H O H O H O + O CAT Fe III CAT Fe IV = O + CAT Fe III ONOO NO ONOO NO + O NO NO

45 Utilization of ROS signaling chemistry for novel, mechanismbased antitumor strategies

46 Tumor cell O CAT CAT O O CAT O O CAT O Inhibition or inactivation of CAT sicat O O O O O O O Intercellular ROS signalling

47 Fibrosarcoma

48 Fibrosarcoma plus Catalase inhibitor

49 Ovarial carcinoma cell line

50 Ovarial carcinoma cell line after catalase inactivation

51 Distinct ways to sensitize tumor cells for apoptosis induction

52 1 Inhibition of catalase by 3aminotriazole O NO ONOOH H + NO OH O ONOO INH Cl Arginase O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O O H O + O +H + +Cl O 3Aminotriazol

53 Scavenger for superoxide anions and inhibitor of NADPH oxidase APOPTOTIC CELLS ( % ) CONTROL TAU ABH MANNITOL MnSOD AEBSF LNAME FeTPPS ,1 6, 1, NO/peroxynitrite pathway 3AT (mm) HOCl pathway

54 SiRNAmediated knockdown of catalase APOPTOTIC CELLS ( % ) WITHOUT INH MnSOD ABH TAU LNAME 0 0 0,64 1,6 4 9,6 4 sicat (nm) sirnamediated knockdown of catalase sensitizes tumor cells for ROS signaling

55 3 Singlet oxygenmediated inactivation of catalase Light O NO PHOTOFRIN Arginase ONOOH H + NO OH O ONOO x O O H O + O + H + O Cl 1 O HOCl + OH 1 O 1 O Arginine Citrulline OH + Cl + O Apoptosis O H O + O +H + +Cl

56 4 Inactivation of catalase by tumor cellderived singlet oxygen 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

57 Cl O HOCl OH + Cl + O POD O + H + H O + O H O H O H O + O CAT Fe III CAT Fe IV = O + CAT Fe III NO ONOO H + NO ONOO NO + O O ONOOH NO + OH NO NO

58 Cl O HOCl OH + Cl + O POD O + H + H O + O H O H O H O + O CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

59 Cl O HOCl OH + Cl + O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

60 Cl O HOCl OH + Cl + O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

61 Cl O HOCl OH + Cl + O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

62 Cl O HOCl OH + Cl + O 1 O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

63 Cl O HOCl OH + Cl + O 1 O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH FASR

64 Cl O HOCl OH + Cl + O 1 O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH NOX1 FASR

65 Cl O HOCl OH + Cl + O 1 O POD O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH NOX1 FASR

66 Cl O HOCl OH + Cl + O 1 O 1 O POD 1 O O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH NOX1 FASR

67 Cl O HOCl OH + Cl + O 1 O 1 O POD 1 O O + H + H O + O H O H O H O + O CAT Fe III NO CAT Fe III CAT Fe IV = O + CAT Fe III NO NO NO NO ONOO H + NO ONOO NO + O O ONOOH NO NO NO + OH

68 Cl O HOCl OH + Cl + O 1 O 1 O POD 1 O O + H + H O + O CAT Fe III NO x CAT Fe III NO NO NO NO ONOO H + O ONOOH NO + OH

69 Cl O HOCl OH + Cl + O 1 O 1 O 1 O POD 1 O 1 O 1 O 1 O O + H + H O + O CAT Fe III NO x CAT Fe III NO NO NO NO ONOO H + O ONOOH NO + OH

70 Cl O HOCl OH + Cl + O 1 O 1 O 1 O POD 1 O 1 O 1 O 1 O O + H + H O + O CAT Fe III NO NO x CAT Fe III More singlet oxygen generation More catalase inactivation Restoration of intercellular apoptotic ROS signaling NO NO NO ONOO H + O ONOOH NO + OH

71 5 Inhibition of catalase by mab sensitizes tumor cells for ROS signaling O NO ONOOH H + NO OH O ONOO mab Cl Arginase O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O Apoptosis O H O + O +H + +Cl O

72 In addition to restoration of ROS signaling, anticat triggers dynamic, automplificatory inactivation of catalase by singlet oxygen (next page) O NO ONOOH H + NO OH O ONOO Cl Arginase O O H O + O + H + O HOCl + OH Arginine Citrulline OH + Cl + O O H O + O +H + +Cl

73 H O ONOO ONOO H O H H O x O CAT ONOO ONOO H O ONOO

74 ONOO H O H ONOO O H O 1 O x 1O CAT ONOO H O ONOO

75 ONOO H O H ONOO O H O 1 O x 1O CAT ONOO H O ONOO

76 H O ONOO ONOO H H O x O CAT ONOO H O ONOO

77 1 O 1 O H O ONOO ONOO H H O x O CAT ONOO H O ONOO

78 1 O 1 O ONOO x ONOO ONOO H O H O H O CAT H O

79 ONOO x ONOO ONOO H O H O H O CAT H O

80 Cl HOCl O NO ONOO H + POD O + H + H O + O OH + Cl + O O ONOOH x CAT NO + OH

81 Cl HOCl O NO ONOO H + POD O + H + H O + O OH + Cl + O O ONOOH x CAT NO + OH Apoptosis

82 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 ROSrelevant phenotype of tumor cells (NOX1 activation combined with expression of membraneassociated catalase) and do not affect intracellular catalase of nonmalignant cells

83 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 proofofconcept experiments in vivo have been successful

84 Summary: Transformed cells generate extracellular superoxide anions Extracellular superoxide anions and their dismutation product H O 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 apoptosisinducing ROS signaling

85 Resistance of tumor cells is acquired through expression of membraneassociated catalase Catalase inhibits HOCl signaling through decomposition of H O and interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite Inhibition of membraneassociated catalase of tumor cells with monoclonal antibodies or its inactivation through singlet oxygen causes ROSmediated apoptotic selfdestruction of the tumor cells This efficient and highly selective principle is a promising approach for novel strategies for tumor therapy

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87 I am grateful to Manfred Saran Reinhold Schäfer Galina Deichman for sharing their concepts, ideas and biological systems

88 Our work was funded by DFG Deutsche Krebshilfe ClottenStiftung RiscRad EuroTransBio Hans Sauer Stiftung