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Doctoral Thesis

Subcellular events in photodynamic therapyrole of mitochondria

Author(s): Klein, Sabine Doris

Publication Date: 2000

Permanent Link: https://doi.org/10.3929/ethz-a-003865505

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Diss. ETH No. 13382

ar events in photo namic

ole of mitochon

A dissertation submitted to the

Swiss Federal Institute of Technology (

for the degree of Doctor of Natural Sciences

presented by

Sabine Doris Klein

Dipl. Natw. FIX

born 13”” August I972

citizen of Illnau-Effretilton, ZH

accepted on the recommendation of

Prof. Dr. I(. H. Winterhalter, examiner

Prof. Dr. C. Richter, co-examiner

PD Dr. H. Walt, co-examiner

2000

Diss. ETH No. 13382

Subcellular events in photodynamic therapy:

role 01" mitochondria


Swiss Federal Institute of Technology (ETH) Zürich

for the degree of Doctor of Natura1 Sciences

presented by

Sabine Doris Klein

Dipl. Natw. ETH

born 13th August 1972

citizen of Illnau-Effretikon, ZH


Prof. Dr. K. H. Winterhalter, examiner



2000

Diss. ETH No. 13382

Subcellular events in photodynamic therapy:

role of mitochondria


Swiss Federal Institute of Technology (ETH) Zurich

for the degree of Doctor of Natura1 Sciences

presented by

Sabine Doris Klein

Dipl. Natw. ETH

born 13th August 1972

citizen of Illnau-Effretikon, ZH


Prof. Dr. K. H. Winterhalter, examiner



2000

Table o F contents

ABLE OF CONTENTS

1. SUMMARY

2. II;USAl\lritlENl~‘~tSSUNC:

3. h3RRFVIA’TIONS

4. INTRODUCTION

4.1. Photo~yrmmic tkmpy

4.1.1. Photosensitiscrs

4.1.1.1 . S,10,15,2O-‘~ctra(~~~-l~yd~o~y~~henyl)clrlori~~

4. I .I 2. Meso-tetra(4-sulfb~~atophenyl)po~~~lli~~e

4.1. I .3. Victoria blue 130

4.1.1.4. &Aminolaevulinic acid

4. I .2. Mechanisms of photosensitiser accumulation

4.1.3. Localisation ofphotosensitisers

4.1.3.1 . Accumulation in tissues

4.1.3.2. Snbcellular localisation

4.1.4. Fluorescence, type I and type II photochcmical reactions

4.1.5. 0sidative damage

4.1.6. Direct and indirect cell killing

4.1.7. Resistance to or due to PDT

4.1 .S. Mitochondria in photodynamic therapy

4.1.8.1. Studies on cells

4.1.8.2. Studies on isolated mitochoudria

4.2. Apoptosis

4.2.1, Apoptotic cascade

4.2.2. Caspases

4.2.3. Mitochondria and apoptosis

42.4. Rcl-2 protein Crnily

42.5. PDT and apoptosis

5. AIMS OF TllE S’I’lJDY

5

7

9

‘1 1

11

II

12

13

14

14

IS

16

16

‘1 6

17

20

21

22

22

23

24

26

26

27

27

28

29

30

2 Table of contents

~~~~~~~~~~~~~~~~~~~~~~

6.1. iWM3?nls

6.1.1. Chemicals

6.1.2. Cell lines

6.2. M~?rllorls

6.2.1. Tsolation ofmitochondria

6.2.2. Incubation ofmitochondria with mTHPC and irradiation

6.2.3. Determination ofprotein oxidation and lipid peroxidation

6.2.3.1. Protein oxidation

6.2.3.2. Thiobat%ituric~ acid-reactive substances

6.2.3.3. Lipid hydroperoxides

6.2.4. .Determination of mitochondrial oxygen consumption

6.2.5. Determination of the mitochondrial membl-ane potential and of Cazl

uptake

6.2.6. Extraction of mTHPC from mouse liver mitochondria

6.2.7. Confocal laser scamping microscopy

6.2.8. Fluorescence microscopy

6.2.9. Irradiation of cells with laser light

6.2.10. Tllumination of cells with white light

6.2.11. Colony formation assay

6.2.12. M’iT assay

6.2.13. Determination of cellular oxygen consumption

6.2.14. Western blot analysis

6.2.15. Nitrate and nitrite determination

6.2.16. Statistical analysis

7. RES~JL,TS

7.1. PJlotosensitisntiorl of isolated mitochotldrin bv nl TIIX

7.1.1. Protein oxidation and lipid peroxidation

7.1.2. Decreased oxygen consumption

7.1.3. Decreased mitochondri al membrane potenti al and Ca3’- uptake

7.1.4. Accumulation of m7’HPC in mouse I iver mitochondria

31

3 ‘1

31

32

33

33

33

34

34

34

3s

35

36

36

37

38

3 8

35

39

39

40

40

41

41

43

43

43

45

47

49

3 Table of contents

7.3. Suhcellulnv events aj?erphotosensitisntion of’MCF-7 cells by mTHPC nnd

irrmiintio~r

7.2.1. Subcellular localisation ofmTHPC

7.22. Colony formation


7.2.4. Decreased mitochondrial transmembrane potential

7.2.5. Effects of antioxidants

7.2.6. Increased intracellular calcium levels and nitric oxide formation

7.3. Protection against md serwitisntiorl to ill \:itr*o photodynamic themJg/ h,,

overexpression qf Bcl-2 7.3.1. Subcellular localisation of VB-HO, mTFTPC and TPPS4 in NV3 and

B22 cells

7.3.2. IJptakc of VB-BO, mTHPC and TPPSd into BV3 and B22 cells

7.3.3. Dark toxicity and light-induced toxicity of VB-BO, mTHPC and

TPPS4 in BV3 and B22 cells

7.3.4. Cytochrome c release, membrane blebbing and annexin V binding in

BV3 and B22 cells

7.3.5. Dark toxicity and light-induced tosiciiy ofhzE in BV3 and B22 cells

7.3.6. Uptake and dark toxicity ofVB-RO in N417 cells

7.4. Hcl-2 protects mitochondria lx/f not ccllsjko7w ceravMide-i~7clz~~ce~l @xt,s

7.5. Sliin photosensitivit), in PDT

7.5.1. Protective substances

7.52. Hyperiforce, a Hypericum extract. displays characteristics of a PS

8. nrscussIoN

8. 1. Mitochonclrin ~7s (I tctrgef c!f‘mT~~~CyJ7otoser2sitiscttion3~~e77sitis~tio~l

S. 1.1. Localisation of mTHPC

8.12. Photosensitisation of isolated mitochondria

S. 1.3. Photosensitisation of MCF-7 cells

8.2. Oxidativc cJa771crge l,l; PI THPC-mediated i77 vitro PD7

8.3. Protecti ngairlst arid smsitisatiou to iit lYf7.0 PDT b.1: ovcre.xpr’essio~l q/

Bcl-2

8.3.1, Enhanced sensitivity of Bcl-2 0Yerespressing 1,920 cells to the lipo-

philic cationic photosensitiser victoria blue BO

51

51

53

54

55

56

57

59

59

60

60

63

66

68

70

72

72

73

76

76

76

77

7s

79

so

so

4 Table of contents

8.32. Effects of ceramide

8.3.3. A2E, a component oflipofixcin, as aphotosensitiser

8.3. Role @Cc? and NO in PDT

8.5. Skin photosensitivity

8.5. I, Protection against skin photosensitivity

8.52. Synergistic effects oftwo photoseusitisers

8.6. Conclusiom

9. REFERENCES

83

83

84

S6

86

57

58

89

103

10s

107

109

5 1 Summary

I. SUMMARY

In photodynamic therapy (PDT), a tumour-localised photosensitiser (I’S) is

excited by visible light, resultin g in the formation of reactive oxygen species and

destruction of cells. The nature of the PS and the experimental protocol determine its

subcellular localisation, and hence the primary target site and following events.

Reactive oxygen species fbrmed upon irradiation are short--lived and react with

biomolccules in their immediate vicinity.

In MCF-7 cells the second-generation PS 5,10,15,20-tetra(/q+-hydroxyphenyl)-

chlorin (m’THPC) localises in the nuclear membrane, plasma membrane and organel les,

some of which are identified as mitochondria by co-localisation with rhodamine 123.

When isolated rat liver mitochondria are incubated with mT?IPC and subsequently

irradiated with light of 652 nm, their lipids and proteins become oxidatively modilied.

In conseyuence, mitochondrial functions including Ca”-!- uptake, respiration and ATP

synthesis are impaired, and the transmembr-ane potential (AY) is decreased. lncuhation

of MCI;-7 cells with mTHPC and irradiation results in decreased viability, oxygen

consumption and AY. One to two hours after irradiation intracellular Ca”-!- levels rise.

This is accompanied by a biphasic formation of nitrite and nitrate. The LIST of

scavengers of reactive oxygen species demonstrates that mTHPC acts GCI type I

(electron or hydrogen transfer) and type II (energy transfer to molecular oxygen

(Tenelating singlet oxygen) photochcn~ical reactions. b

Cells with increased A\-Y, e. g., carcinoma cells or L,929 cells overexpressing

Bcl-2, are protected against apoptosis and take up higher concentrations of lipophilic

cationic compounds. Therefore, positively charged I’S should preferentially accumulate

in these cells and permit selective destruction. The sensitivity of control (BV3) and

Bcl-2 ovcrexpressing (B22) L929 cells to three, charged and uncharged, PS alone or in

combination with light was investigated. In both cell lines, victoria blue BO (VB-BO;

positively charged) localises in mitochondria, mTHPC (uncharged) in membranous

structures and meso-tetra(4-sulfonatophenyl)l~or~~~i~~e (‘CPPS~; negatively charged) in

lysosomes. Uptake of mTHPC and TPP& into both cell lines is equal, whereas B22

cells take up more VB-BO than BV3 cells. They, consequently, display higher

sensitivity to this PS both in the dark and after light exposure. Dark toxicity of TPPSJ

6 I Summary

and mTHPC, and light-induced toxicity of mTHPC are equal in both cell lines.

Incubation with TPP& and illumination with white light is more toxic to BV3 cells.

After incubation with these PS and illumination cells show signs of apoptosis including

the release of cytoc.hrome c from mitochondria, membrane blebbing, enhanced annexin

V binding and rounding off. Overexpression of Bcl-2, which protects against apoptosis-

inducing stimuli, renders B22 cells more sensitive than control cells to the toxicity of

VB-HO, a lipophilic cationic PS. This provides a potential strategy for cancer cell

targeting in vivo.

A m+jor side effect of PDT is skin photosensitivity caused by accumulation of

PS in the skin. Protection of patients cannot be achieved by sunscreens, but only by

creams that do not allow penetration of visible light. Cover creams containing pigments,

c. g., Ti02 and FclO?, show ir? I&O efficacy to reduce light-induced cell killing of

human keratinocytcs incubated with mTHPC. A Hypericum extract, used in the

treatment of depression, also contains compounds that accumulate in the skin of patients

and display characteristics of a PS. Synergistic effects of Hypericum perforatum extract

with S-a~~ninolaevulinic acid, a PS precursor, are observed iti vitro and ill vivo.

Incubation of human keratinocytes with both compounds in the dark or followed by

light exposure results in higher cell killing than expected for additive toxic effects.

7 2. Zusammenfassung

2. USAIWMENFASSUNG

Bei der photodynamischen Therapie (PDT) wird ein Photosensibilisator (PS),

der sich im Tumor angereichert hat, tnit sichtbarem Licht angeregt. Dies erzeugt

reaktive Sauerstoffspezies, welchc die Zellen zerstiiren. Die subzellul%re Lokalisation

eines PS ~md folglich such das primgre Ziel und nachfolgendc Ereignisse h%ngen von

seiner Natur und der Versuchsanorctuuilg ab. Die durch Bestrahlung entstehenden reak-

tiven Sauerstol-fspezies sind kurzlebig und reagieren mit Biornolektilcn in ihrer mimit-

telbaren IJmgcbung.

Der Zweitgcnerations-PS 5, 10,15,20-Tetra(nl-hydroxyphenyl)chlorin (mTHPC)

reichert sich in Kernmembran, Zellmembran und in Organellen von MCF-7 Zellen an.

Einigc dieser Organellen konnten als Mitochondricn identifiziert werden, da sie sic11

gleichzeitig such mit Rhodamin 123 anf%rben liessen. Inkubation isolierter Rattenleber-

mitochondrien mit mTHPC und anschliessende Bcstrahlung mit Licht von 652 nm

modifiziert ihre Lipide und ‘Proteine oxidativ. Mitochondriale Funktionen einschliess-

lich Ca2’-Aufilahme, Atmung utld ATP-Synthese sind in der Folge beeintr%chtigt, und

das transmembrane Potential (AY) ist verringert. Inkubation von MCF-7 Zellen mit

ITITI-TIT und Bestrahlung haben verminderte Viabilitat, Sauerstoffverbrauch und AY

zur Folge. Eine bis zwei Stunden nach Restrahlung steigen die intrazellul%ren Ca2’--

Konzentrationen. Dies ist von einer zweiphasigen Bildung von Nitrit und Nitrat

begleitet. Einsatz von Substanzen, die reakti\,e Sauerstoffspezies abfangen, ergab, class

mTFTPC photochemische Reaktionen vonl Typ 1 (Elektronen- oder Wasserstofc-

iibertragung) und Typ II (Energietibertragung auf molekularen Sauerstoff ZLII‘ Bilchmg

von Singulett-Sauerstoff) eingeht.

Zellen mit erhiihtem AY, z. Bsp. Karzinomzellen oder Bcl-2 iiberexprimierendc

L929 Zellen, sind gegen Apoptose geschiitzt und nehmen hijhere Konzentrationen

lipophilcr kationischer Substanzen auf. Deshnlb sollten positiv geladene PS vorzugs-

weise in diesen Zcllen akkumuliercn uncl eine selektivc Zcrst6rLmg ermiiglichen, Die

SensitiviGt von Kontrollzellen (RV3) unci Bal-2 iiberesprilnie~.enden (1322) L929 Zellcn

auf drei, geladene und ungeladcnc. PS allein oder in Rombination rnit Licht wurde

untersucht. In beiden Zelllinien ist Victoria Blau HO (VB-BO; positiv geladen) in

Mi tochondrien, m’THPC (ungeladell) in Merl~branstrukture~l und Meso-t-etra(4

8 2. Zusammenfassung

sulfonatophenyl)porphin (TPPS4; negativ geladen) in Lysosomen lokalisiert. Die

Aufnahme von mTHPC und TPPS4 in beide Zelllinien ist vergleichbar, w&end B22

Zellen mehr VB-BO als BV3 Zellen aufilehmen. Deshalb reagieren B22 Zellen sowohl

in Dunkelheit als au& bei Lichtexposition empfindlicher aufdicsen PS. Dunltcltoxizit%t

von m’THPC mrd TPPS4 und Licht-induzierte ToxiziGt von mTHPC sind in beiden Zcll-

linicn gleich. BV3 Zellen reagiercn emp6ndlicher auf Inkubation mit TPPS4 und

Belcuchtung mit weissem Licht. Nach Inkubation mit I’S und Beleuchtung zeigen die

Zellen Anzeichen von Apoptose, einschliesslich der Freisetzung von Cytochrom c aus

Mitochondrien, Bl&chenbildung an der Zellmembran, erhiihte Bindung von Annexin V

sowie Abrundung. Die I%erespression von Bcl-2, welche die Zellen gegen Apoptose-

induzierende Substanzen schiitzt, ma&t die B22 Zellen empfindlicher fir die toxische

Wirkung von VB-HO, einem lipophilen kationischen PS, als BV3 Zellen. Dies criiffilet

tine miigliche Strategie fiiir das Erfassen von Krebszcllcn irz IGVO.

Ein schwcrwiegender Nebeneffekt dcr PDT ist die infolge Ansammlung des PS

in der Haut hcrvorgerufene Hautempfindlichkeit auf Licht. Sonnencr&ne ltann keincn

ausreichendcn Schutz ftir Patienten bieten, sondern nur Cr&mes, die das Durchdringcn

von sichtbarem Licht verhindem. Abdeckcr&mes mit Pigmenten wie TiO2 und FclC+

reduzieren in vitro den Licht-induzierten Zelltod tncnschlicher Keratinozyten, die mit

mTHPC inkubiert worden sind. Ein Hypcricum-Extrakt, der I% die Behandlung von

Depressionen verwendet wird, enth5lt Kompotlenten, die sich in der Haut von Patienten

anreichern und Eigenschaften von PS aufweisen. Synergistische Effekte des Hypericum

perforatum-Extraktes mit 6-Aminol~ivulins~~~r~. cinem PS-VorKufer, werden i?l vitro

und in vivo beobachtet. lnknbation von menschlichcn T<eratinozyten mit beiden

Substanzcn, im Dunkeln oder get‘olgt von Lichtexposition, rcsultiert in h6herem Zelltod

als fir additive toxischc Wirkungcn erwartet wiirde.

9 3. Abbreviations

3. ABBREVIATIONS

AA

ALA

arsenazo III

Asc/TMPD

AV

BHT

C2 ceramide

C6 ceramide

CCCP

CF

CLSM

DHC

DHR

DMF

DMSO

AYJ

EDTA

EGTA

FCCP

FCS

FITC

Hepes

HpL)

LDL.

LII

LOOH

mTHPC

MTT

antimycin A

6-aniillolaevulillic acid

3,6-bis~(2-ause~~opl~er~yl)a~o]-4,5-dil~ydroxy-2,7-na~l~t~~alene-

di sulfoni c acid

ascorbate/tetramethylphenylenediaminc

annexin V

butylated hydroxytoluene

N-acctylsphingosine

N-hexanoylsphingosine

carbonylcyanide-3-ch1orophenylhydrazone

colony formation

confocal laser scanning microscopy

C2 dihydroceramide

dihydrorhodaminc 123

dimethyl fkran

dimethyl sulfoxide

mitochondrial transmetnbrane potential

ethylenedinitilotetraacetic acid

ethylene glycol bis(P-aminoetllylcther)-N,N,N’,N’-tetraaccti~ acid

carbo~~ylcyanide-4-trifluoron~etl~oxypl~enyll~ydrazone

foetal calf serum

-Iluorescein isothiocyanate

4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid

haematoporphyrin derivative

low density lipoprotein

unsaturated lipid

lipid hydropcroxide

5,10,15,20-tetra(nz-l~ydroxy~l~enyl)chlol-in

3-~4,5-din~etl~yltl~iazol-2-yl]-2,5-diphenyltetrazoliun~bl-omide;

thiazo lyl blue

10 3. Abbreviations

NOS

PDT

PI

PPlX

PS

RI11 23

SDS

TR AR S

TPPS.,I

VB-HO

nitric oxide synthase

photodynamic therapy

propidium iodide

protoporphyrin TX

photosensitiser( s)

rhodamine 123

sodiumdodecylsulfate

thiobarbitmic acid-reactive substances

meso-tetra(4-sulfonatopl~eiiyl)porphine

Victoria blue BO

4. Introduction

Photodyrinmic therapy (PDT) is based on light activation of a photosensitiser

(PS) that has accumulated at a target site, e. g.- tumour tissue, resulting in the formation

of reactive oxygen species and, finally, cell death (for reviews, see Fisher el cll., 1995;

Henderson and Dougherty, 1992). The basic steps of PDT are sutmnarised in Table 1

and will be discussed in the following paragraphs.

1.

2.

3.

4.

5.

6.

7.

8.

9.

Systemic or topical application of the PS

Accumulatioll/l-ete~lti01i 0 f the PS in the target tissue (e. g. tumour)

Photoexcitation: penetration and absorption of light

Intersystem crossover to triplet state of the PS

Gcncration of reactjve intermediates (radicals, reactive oxygen species)

Photooxidation of cellular constituents (proteins, lipids, nucleic acids)

Functional and structural alterations (enzyme activities, membrane fluidity, mutations)

Ccl1 damage/death: necrosis or apoptosis (primary cell death)

Vascular occlusion, inflammation (secondary cell death)

PDT is Ltsed to treat a variety of dysplasias and superficial cancers, i. e.,

gynaecological tumours, skin metastases of mammary carcinoma, cutaneous, l~mg,

oesophagus and bladder cancer, and cervical and vulva1 intraepithelial neoplasias

(Fisher et nl., 1995).

4.1.1. Photosemitisers

PS are non-toxic substances that accumulate in the target tissue (CL g. tunioW)

and absorb light in the visible (preferably abovc 600 nm) or infrared range of the

12 4. Introduction

spectrum. At the beginning of PDT, iu the 195Os, a mixture of porphyrins

(llae~~latopor~~llyrin derivative, HpD) was used as PS. The most active components were

identified as dihaematoporphyrin ethers and esters. Commercially available preparations

(photofiin” and photofrin II’@) contain mostly porphyrin dimers aud oligomers. ‘The

drawbacks of a variable and complex mixture, low absorption above 600 nm and

prolonged cutaneous sensitivity led to the development of new, so called ‘second-

generation’ PS (Fisher et nl., 1995). The ideal properties of a PS are defined as:

chemical purity, minimal dark toxicity, high tumour selectivity, large absorption

coeff?cient at a high wavelength, hi,oh photochemical reactivity, and high ability to

induce (in)direct cell killing (Penuin g and Dubbeltnan, 1994). Absorption at a high

wavelength is preferable, since light above 600 nm can penetrate tissues better due to

lower absorption by water, hacmoglobin etc.

PS can be classified, e. g., by their chemical structure, and include porphyrins,

chlorins, bactcriochlorins, purpurins and plitlialocya~~ilines. As examples, chemical

structures of PS used in this thesis are shown in Fig. 1. Also natural compounds, like a

Hypericum extract used in the treatment of depression, may have photosensitising

properties (Golsch et nl., 1997; Remd et al., 1999). Photodynamic effects are also

observed in diseases like porphyria which results from impaired haem synthesis (Ihr

reviews, see Elder et ni., 1997; Moore, 1998), or are presumed to take place, e. g., in

age-related macular degeneration (Gaillard et nl., 1995; Wihhnark et al., 1997).

4.1.1.1 . 5,10,15,20-‘I’etra(nz-l~ydroxyphenyl)chloriu

5,10,15,20-Tetra(nl-hydroxyphenyl)chlorit~ (m?XPC, temoporfin, Foscan@) is a

neutral lipophilic second-generation PS ITit a high absorption at 652 nm. It

inhomogenously localises in the cytoplasm around the nucleus of MCF-7 and V79 cells

(Hornung et nl., 1997). When cells are incubated with mTHPC and exposed to light,

they are more sensitive at pH 6.8 than at pW 7.2 although the level of drug uptake is pH-

independent (Ma et nl., 1999). Compared to tetra( ~~~-l~ydroxypbe~~yl)porphi7ne aud

photofrin II”@, incubation Lvith rn’l‘HPC mcl irradiation more efficiently induces cell

death when a wavelength abo~~c 600 nm is used to excite the I’S (Ma et LLI., 1994). In i/l

vI‘vo studies in mice bearing mammary carcinoma, tumour destruction by PDT with

mTHPC was more efficient than with the cot-responding porphyrin (Peug et (II., 1995).

13 4. Introduction

Clinical trials with mTHPC-mediated PDT, e. g., for oral cancer (Fan et nl., 1997) and

sy~tan~o~~s cell carcinomas of the oesophagus (Savary et nl., 1998), are ongoing.

,, J’ --.

Fig. 1. Chemical structures of 5.10:15,20-t-etra(nl-1rydroxypheilyl)chlol-i (mTFIPC). meso-tetra(4-

sulfonatophenyl)pot-~liitie (TPP&), victoria blue I30 (VU-BO), fi-amiiiolaevulinic acid (ALA), and

p~otopol-phyriu IX (PPlX).

peso-tetra(4-suIfonatophenyl)l?ol-phine (TPPSJ is anionic and hydrophilic, and

localises in lysosomes (Malik t?f ill., 1997). Normal lcukocytcs are less sensitive to

TPPSd-mediated phototoxicity than HLGO and HEL leukaemia cells (GrcbenovA et nl.,

1997). It is, however, 50-fold less efficient as a PS than is pliotofrin II@ in WiL>r cells

14 4. Introduction

exposed to 10 pg/ml of either drug (West and Moore, 1989). Ivr vivo, TPPS4 has been

used as a PS for PDT of cutaneous metastases of breast cancer where it was applied

locally to avoid skin photosensitisation (Lapes et nl., 1996).

4.1.1.3. Victoria blue BO

Victoria blue BO (VB-HO) is cationic and lipophilic, and is taken up by

mitochondria (Modica-Napolitano et al., 1990). Incubation of isolated mitochondria

with VB-BO induces uncoupling of oxidative phosphorylation, and when followed by

irradiation, it inhibits complex I of the respiratory chain (Modica-Napolitano et al.,

1990). VH-BO has both a much higher dark toxicity and light-induced toxicity than

TPPS4 in human leukemic cell lines K-562 and TF-1 (Fiedorowicz ef NL., 1993).

Monocytes and leukemic cells are more sensitive than lymphocytes to VB-BO-mediated

phototoxicity, suggesting a possible application of VB-BO for selective depletion OC

monocytes or sensitive leukemic cells (Fiedorowicz et al., 1997). A series of

con~po~mds based on the structure of VB-BO 1~~~s synthesised, but none of them

equallcd toxicity of VB-BO in the dark or with illumination (But-row et al., 1995).

4.1.1.4. K-Aminolaevulinic acid

~-A1llinolaevulinic acid (ALA) is not a PS but a prodrug (Gibson et al., 1997).

ALA is formed from glycine and succinyl-CoA in the first step of the synthesis of

haem. When added to cells, it enhances the formation of protoporphyrin IX (PPIX) and

haem (for review, see Peng et crl., 1997). PPIX accumulates in the mitochondria and can

serve as a PS because of the limited capacity of ferrochelatase, which incorporates iron

into PPIX to form haem. In some tumours, the activity of porphobilinogen deaminase,

another enzyme in the haem synthesis pathway, is increased and that of ferrochelatase

decreased, so that PPIX accumulates preferentially in tumour cells (van Hillegersberg ei!

al., 1992; Hinnerl ef crl., 1998). Porphobilinogen deaminase activity increases when

tumour cell lines are incubated with ALA Dale to tlci /!01’0 synthesis of the enzyme

(Gibson et nl., 199s). Iron chelators, e. g., etl~ylcncdialninetetraacetic acid (EDTA) and

dcsferrioxamine, enhance PPIX accumulation in cells exposed to ALA (Berg et al.,

1996). Its major advantage compared to other PS is that ALA can be applied topically

without the side effect of cutaneous photosensitivity, its disadvantage is a low light

absorption of PPIX at wavelengths above GO0 nm.

15 4. Introduction

41.2. Mechanisms of photosensitiser accumulation

Hydrophilic PS arc transported in the blood Gn albumin and globulins

(Villanueva and Jori, 1993), and localise in the stroma of ~UIJ~OLK and other tissues

(Fisher et nl., 1995). Hydrophobic PS can be incorporated into plasma lipoproteins,

e. g., low density lipoproteins (LDL), and arc taken LIP by cells partially via reccptor-

mediated endocytosis (Allison et 01.) 1994; Soncin et al., 1995). I/? vilro and ilz vim,

m’THPC binds to LDL and HDL (Michael-Titus ef <rZ,, 1995). Higher selectivity in

tumour targeting could be achieved by associating the PS before injection with delivery

vehicles, e. g. LDL, which can interact preferentially with tumour cells (for review, see

Reddi, 1997). Cells with a high content of LDT., receptors, such as mitotic cells,

including tumour and endothelial cells would preferentially take ~tp the PS.

Another strategy OC targctin, 0 consists in antituniour monoclonal antibodies as

carriers for the PS. Turnour selectivity of antibody-conjugated mTHPC is higher in

comparison with t-ret mTHPC (Vrouenraets et nl., 1999). 171 vitro, the efficacy of

mTIJPC is higher when coupled to an interualising rather than to a noninternalising

antibody (Vrouenraets et nl., 1999).

Lipophilic cationic PS are taken LIP into cells in response to the cellular and the

mitochondrial transmembrane potential (AY). Carcinoma cells with their elevated AY

(Nadakavukaren et nl., 1985) are sensitive to positively charged compounds, e. s.,

rhodamine 123 (Rh123) (Lampidis et al., 1983: Davis et nl., 1985). The cause of this

increase in AY, its relation to cancer and to resistance to apoptosis are not fully

understood, however, this feature of tunlour cells can be exploited therapeutically, i. e.,

by targeting them with cationic drugs (Sun et al., 1994; Roya et al., 1996; Nocentini et

nl., 1997). Also in GVO, a cationic phthalocyanine is a very potent PS (Cruse-Sawyer et

al., 1998).

16 4. Introduction

41.3. Localisation of photosensitisers

4.1.3.1. Accumulation in tissues

To determine optitnal time laps before irradiatioa of the target tissue after

injection of the PS (drug-light intcrvnl)i PS le\rels in turnour and normal tissues are

determined either by fluorescence measurements or by extraction and HPLC analysis. In

order to destroy tumour and spare normal tissues, a time point is chosen with a high

t~mour to normal tissue ratio of PS concentrations. For many clinically used PS this is

between 24 and 72 h after injection. 4 fluorescence study of mTHPC in malignant and

normal tissues in rats revealed high accumulation of the PS 24 h after injection in

turnour tissue, small intestine, lung and liver. Forty-eight h after injection, highest

mTHPC fluorescence was found in turnour tissue. PS concentrations in tumour tissue

were always higher than in the surrounding muscles (Alian et al., 1994). In mice

bearing mammary carcinoma, concentrations of m”lXPC peaked at 24 to 48 11 after

injection, and only liver, urinary tract and skin took up more mTHPC than those

tmiours (Peng et al., 1995).

4.1.3.2. Subcellular localisation

Subcellular localisation of a PS is detertnined either by con focal laser scanning

microscopy using the fluorescent properties of the PS or by fractionation of cells,

differential centrifugation and HPLC analysis. In b ~~eneral, localisation depends on

hydrophobicity and charge of a PS (Table 2; Woodburn et 01.~ 1991).

The intracellular localisation of a PS deternGnes also the sites of danlage upon

irradiation (for review, see Pcng et nl., 1996). Interestingly, Woodburn et nl. (1992)

found that porphyrins localised in lnitochondria displayed higher phototoxicity than

porphyrins with lysoson~al or difhlse cytoplasnGc localisation

17 4. Introduction

Table 2. Localisation of a PS depends on its hydrophobicity and charge.

hydrophobic

hydrophobic

hydrophi li c

hydrophilic

cationic

anionic

cationic

anionic

cytop lasmic perinuclear 11

mitochondri a a, c, cl

diffilse cytoplasmic ”

nucleus c, r

diffuse cytoplasmic plus lysosomes a

a Woodburn et nl., 1991; ' Honm~g ef al., 1997 (m’THl’C); ’ Johnson ef 01.) 198 1; cl

Modica-Napolitano

et nl., 1990 (VU-RO): ’ Villanucva et nl., 1992 (meso-tet-~a(4~-~~letl~ylp~idyl)pol~~~iiie); f Riini~ ef ni.,

19% (metbylene blue); ’ Malik e;t nl., 1997 (TPPS.+)

4.1.4. Fluorescence, type I and type 11 photocl~ernical reactions

Light absorption excites the PS fioni the ground state to the hrst singlet state

(Scheme I). It can decay back to its ground state by emitting light (fluorescence) which

is useful for tumour localisation as well as for subcellular localisation of the PS itself.

i vs

SO

Sche~mc 1. Jablonski diagram. Upon absorption of a photon, a PS in the pound slate So is excited to S,

state. It n~ay decay back to the gwnd state by enlission of light (fluorescence) or undergo intersystenl

crossover to a triplet state TI from where light can be emitted in the fern? of phosphorescence.

vr, vibrational relaxation; isc, intersystem crossover, SO, ground state; S, first excited singlet state; T,,

first excited triplet state.

18 4. Introduction

Iutersystem crossover of the singlet PS (‘PS) yields a triplet PS (3PS), which can emit

light (ptlospllorescer7ce), undergo type I or type II photochemical reactions (Scheme 2).

photosensitiser

excited photosensitiser ‘PS

fluorescence radicals electron or hydrogen

transfe1

singlet oxygen 2

localisation studies oxidative damage of proteins, lipids, DNA

Scheme 2. Summary of events following absorption of light by a 13. The excited PS may emit

fhorcscence or undergo type I ox type II photoclmnical reactions. For details, see text,

Type I photochemical reactions are characterised by electron or hydrogen atom

transfer from a substrate, e. g., an unsatmated lipid (LH), to the 3PS (Girotti, 1990)

“PS -k- LR -> ps’- ,- I,” + Hi-

The radical 1;” may then react with 02

L,” -f 02 -> LOO’

In the presence of sufkient amomts of 02 and LH and in the absence of

antioxidants, a chain reaction will OCCUT resulting in the formation of lipid hydro-

peroxides (LOON). The PS’- radical can also react with 02 forming superoxide anion

(,W

ps*- -I- (1, . . ,-> PS f 02’”

Disrnutation of 02*- leads to the fomation of hydrogen peroxide (HQ) and 111

the presence of a Fenton reagent (e. g. Fe”.) also to tzyclrosyl radical (OH”).

19 4. introduction

711 type IT photochemical reactions the 3PS transfers its energy to 302

+s + -+& -“-“--+ ‘13 -t ‘0 2

‘02 -:- LH ,----+ LOOH

‘To distinguish between type I and type 11 photochemical reactions, scavengers or

traps of reactive intermediates of these reactions can be used (Girotti, 1990). An

overview is given in liable 3. However, many of these scavengers lack absolute

specificity, e. g., ‘02 interceptors are also OH’ traps. Tt sho~tld also be considered that

fbr topological reasons a hydrophilic 01-f’ trap may be inefficient against OH” generated

in membranes.

superoxide dismutase

catalase

iron chelators (6~. g. ED’TA) inhibit the formation

mannitol, deoxyribose, butylated hydroxytoluene, a-tocopherol

azide> dimethyl hIran, histidine, p-carotene (no known enzymatic, scavenger)

II20 increases ’ 02 li Mime and accelerates reactions where ‘02 1s an intermecliate

20 4. Introduction

4.1.5. Oxidative damage

The formation of reactive (oxygen) species in type 1 and type II photochemical

reactions results in oxidative damage of proteins, lipids and DNA (Scheme 2). Since the

reactive oxygen species thus generated have a short lifetime and diffusion distance -

singlet oxygen is assumed to diffuse less than 0.07 pm in cells before it reacts (Moan,

1990) - the localisation of the PS determines the topology of damage.

In order not to introduce i%rther mutations in the DNA, PS are preferred that do

not localise in the nucleus. Holveter, many lipophilic PS also accumulate in the nuclear

membrane and may ca~~rse oxidations in DNA close to the membrane. DNA damage by

PDT using HpD or methylene blue but not m’IXPC was detected by means of a cornet

assay (McNair et ~1.) 1997). In vitro, cationic porphyrins are known to have a high

affinity to DNA and induce oxidations and strand breaks when irradiated (Nicotera et

nl., 1994; Mettath cl GIL., 1999). When ceils are treated with either PDT or x-rays with a

dose that induces equal cell killing, x-rays are more mutagenic (Evans et al., 1997;

Paardekooper et 01.) 1997).

Oxidativc modifications of proteins can be assessed by measuring the content of

carbonyl groups. Carbonyl gro~qx are reduced to the corresponding alcohol by NaB3Hli,

and a stable radioactive label is introduced into the protein. Oxidative modifications in

lipids can be either aldehydes (thiobarbituric acid-reactive substances, TBARS) or

LOOH. Tn order to determine whether type T or type II photochemical reactions have

occurred, assays for TBARS and LOOH should be performed. TRARS are end-products

of ii-ee radical peroxidations and should, theorctic,ally, not be generated in pure type II

reactions. LOOH as deternlined by a iodometric assay may or may not be end-products

of photochetnical reactions depending on the conditions, e. g., when ferric iron is

removed by chelation or reductants are absent, LOOH will accumulate. In summary, in

a type I reaction THARS, and in a type II reaction accumulation of LOOH should be

observed (Girotti, 1990).

Apart fioin being probes for mechanistical studies, antioxidants can reduce the

oxidativc damage induced by PDT. They protect red blood cells lrom aggregating alter

PDT for virus inactivation (Ben-Hur et nl.. 1997), and ascorbate protects coagulation

21 4. Introduction

factors from oxidation during photodynamic virus inactivation treatment of plasma

(Parkkinen cf nl., 1996).

Upon PDT, enzymes like glutathione peroxidase, superoxide dismutase or

catalase can be induced in cells (tiadjur ef 01.. 1996), and may possibly lead to a

resistance to PDT.

4.1.6. Direct and indirect cell killing

The major events that iin;1lly lead to cell death as a consequence of PDT are not

_cUlly understood, and may depend on the PS and protocol used.

In vitro, direct cell killing by oxidative damage is observed. .Following PDT with

lipophilic PS, damage to membrane-associated receptors and transport systems, and

disturbance of membrane integrity and fluidity has been reported (Girotti, .I 990).

Research has focused on damage to mitochondrial enzymes. since lipophilic porphyrin

PS preferentially tocalise in mitochondria (see below). Hydrophilic PS localising in

lysosomcs may cause disruption of these organelles resulting in the release of hydrolytic

enzymes into the cytoplasm and ensuing cell death (Brunk et nl., 1997). As discussed

above, damage to DNA is believed not to be significant in PDT-induced cell death.

1~ I:ivo, however, indirect killing due to vascular damage is assumed to play a

major role (Car review, set Fingar, 1996). Vascular occlusion can be induced rapidly

upon irradiation of tissue shortly after drug injection when levels of circulating PS are

high. Within the first minute of irradiation, a decreased oxygen pressure was detected in

marine radiation induced fibrosarcon?as, which was reversible if irradiation was

interrupted (Sitnik et r7/., 1998). Five minuies after ptlotofrin-nledi;ed PDT, the

percentage oi‘ tumour vascular periilsion was decreased to less than 30 %, inducing

hypoxia in tumours (van Gecl et OZ., 1996a). One to 4 II after PDT, reduction of partial

pressure of oxygen is observed in tumours (Reed cl’ NI., 1989). In hypoxia, cells are

protected from further PDT damage by the oxygen limitation of the photodynamic

processes. Protocols with fractionated illumination hat-e been developed (van Gee1 c/

crl., 1996b; 1Mtiller et nl., 199s) in order to address this problem. Depending on the

duration of vascular occlusion, ischemic death and ischelnia-reperfirsion injury may

22 4. Introduction

take place (Cruse-Sawyer et al., 1998), followed also by inflammatory reactions and

phagocytosis of tumour cells (Krosl et al., 1995; Korbelik, 1996).

4.1.7. Resistance to or due to PDT

One major advantage of PDT over other treatment modalities is that it can be

safely repeated several times ) generally without inducing resistance to PDT in contrast

to chemotherapy. Radiation-induced fibrosarcoma cells were subjected to 8 cycles of

photofrin-mediated PDT, \vhich resulted in only 1 .%fold resistance (Sing11 et cd.? 1991).

The PDT-resistant cells were found to produce more ATP, have higher succinate

dehydrogenase activity, and changes in mitochondrial structure compared to cells that

were not PDT-treated (Sharkey Ed nl., 1993). Cross-resistance to cis-diammine-

dichloroplatinum (11) (1.6-fold) but not to adriamycin was reported (Moorehead et nl.,

1994). Chinese hamster ovary multi-drug resistant cells were also resistant to PDT

possibly because they accumulated less PS (Singh et nl., 199 1). When cells were

exposed to oxidative stress induced by PDT and subsequently incubated with

adriamycin, a time-dependent decrease in adriamycin toxicity, correlated with ATP

depletion and cell cycle changes, was observed (Fisher et uZ., 1993). A study with

mTHPC’-mediated PDT did not induce resistance to chemotherapy, radiotherapy or PL)T

in MCF-7 cells, indicating that PDT can be combined with other treatment modalities

(Hortlung et ul., 19%).

4.1.8. Mitochondria in photodynamic therapy

Most studies on PDT effects on mitochondria were done by Salet and Moreno

using HpD or photofi-in as PS (for review, see Salet and Moreno, 1990). By

lluorescence microscopy it was shown that porphyrins accumulate in mitochondria

(Schncckenburger and WustroLv, 1988) afier 24 h inc,ubation time. Microirradiation of

mitochondria but not of the nucleus or hyaloplasm leads to the death of cells incubated

with HpD (Moreno and Sal&, 15X35). When rats were injected with haematoporphyrin,

23 4. Introduction

the PS accumulated in mitochondria of hepatocytes (Cozzaui et nl., 1981). Also when

the endogenous synthesis of porphyrins is stimulated by the addition of ALA,

mitochondria are the prime target (Peng et nl., 1997).

Several mechanisms may account for accumulation of PS in mitochondria. One

major determinant is the partition coefficient: only lipophilic compounds are taken up

by mitochondria, hydrophilic ones are localiscd in lysosomes (Woodburn et nl., 199 1).

There is also evidence that porphyrins bind to benzodiazepine receptors located ou

mitochondrial outer membrane (Verma et al., 1987; Vema et nl., 1998). Positively

charged lipophilic compounds are taken LIP into tnitochondria in response to ilY

(.IdlnSOl et al., 1981).

4.1.8.1, Studies on cells

Upon irradiation, ultrastructural changes in mitochondria are observed: the

organelles appear swollen with a loss of cr-istac (Modica-Napolitano et nl., 1996;

Cernay and Zinmcnnann, 1096). In cells resistant to photo frill-mediated PDT,

morphological changes in rnitochondria were detected (Sharkey et nl., 1993). When

cells were photosensitised with HpD and irradiated. a decrease in cellular ATP levels

paralleled by loss of viability took place (Hilf et nl., 1986). To identify the site of

damage, iodoacetatc, an inhibitor of glycolysis, or oligomycin, an inhibitor of the

mitochondrial ATPase, were added during the experiments. Only iodoacetate had an

additive effect with PDT ou ATP levels, indicating that haematoporphyrin and light

impaired mitochondrial oxidative phosphorylation (Hilf et nl., 1986). Inhibition of

enzymes in cells of a R3230AC matnnmy carcinoma incubated with photofrin II@ and

illuminated was determined (Gibson ef ill., 1989). Pyruvate kinase, a cytosolic enzyme

was not inhibited, in contrast to mitochondrial enzymes. The following order of

inhibition was found: cytochrome c oxidase > l!~F, ATPase > succinate dehydrogenase

> NADH dehydrogenase. Similar inhibition was found with HpD as PS (Hilf cf al.,

1984). Merocyaninc 540, a compound that is preactivated by light before

administration, also af*fects mitochondrial ultrastructure, Rh123 uptake and oxygen

consumption in MCF-7 cells (Gulliya et rrl., 1995). When a porphyrin PS is coupled to

boron atoms, boron neutron capture therapy can be combined with PDT. I/? llitro and j/7

Gvo, such a boronatcd porphyrin cornpound 1r.a~ localised iu mitochondria of glioma

cells (Hill et nl.? 1092). Only timctional mitochondria are a target of this compound,

24 4. introduction

since it was neither cytotoxic nor phototoxic in cells lacking mitochondrial DNA

(Munday et nl., 1996).

Earlier studies revealed the high sensitivity of carcinoma cells compared to

normal cells to lipophilic cationic compounds, e. s. Rhl23 (Lampidis et nl., 1983). ‘This

is a co~~seq~~cnce of elevated mitochondrial and plasma membrane potentials of

carcinoma cells (Davis et nl., 1985) that progressively accumulate dyes like R11123 in

contrast to, e. g., cpithclial-derived cell lines, that equilibrate with the extracellular dye

(Nadakavukaren et nl., 1985). Many rhodamine and cyanine dyes have been evaluated,

the most effective of which was a cyanine with delocalised positive charge (Oseroff el

nl., 1986). The rhodacyanine MKT-077 kills carcinoma cell lines but not an epithelial

cell line. It prolonged survi\~al of‘ nude mice bearing melanoma and reduced growth of

carcinomas (Koya cf al., 1996). Irradiation enhances the mitochondrial toxicity of

MKT-077 (Modica-Napolitano et nl., 199s). When injected into rats, MKT-077 causes

reversible impairment of mitochondrial respiration (Weisberg et nl., 1996). But not all

cationic lipophilic compounds localise in mitochondria. When the negative charge is not

delocalised as in a monocationic porphyrin, the PS does not enter the cells but

accun~ulates at the surface membrane, and irl t*ij~ mostly shows vascular damage rathel

than direct tumour cell kill (Kessel et nl., 1995).

4.1.8.2. Studies on isolated mitochondria

Reduction of mitochondrial fLmctions following PDT of isolated mitochondria

has been extensively studied. Not all fLuunctions arc equally sensitive, the following order

of events with increasing deuteroporphyrin concentration or light dose was found:

uncoupling, dissipation of A’?, inhibition of respiration, swelling and disruption of

mitochondria (Sandber g and Romslo, 1980). Inner rnitochondrial membrane enzymes

are more sensitive to PDT damage than enzymes of the outer membrane, matrix or

intermembrane space (Salct and -Moreno, 1990). Damage to the adenine nuclcotide

translocator is the main cause of oxidative phosphorylation impailment when

mitochondria are exposed to porphyrins and light probably due to oxidation of its thiol

groups (Atlante et ~2.) 19S9). The mitochondrial pemeabil ity transition pore can also be

a target of llael~~atoporl~hyrin-lnediated PDT (&let et u!., 1997).

Cationic PS may show effects on mitochondria evctl in the absence of light.

Cyanines inhibited coupled respiration \\ith /i-h?ldroxybutyrate as substrate in a

25 4. Introduction

concentration-dependent manner, but had no effect on uncoupled mitochondria

(Conover and Schneider, 1981). When isolated mitochondria were incubated with

R11123 in the dark, the respiratory control ratio, ADP/ATP exchange and phosphate

uptake were diminished, These effects were aggravated by irradiation (Atlante et OZ.,

1992).

IJnexpectedly, also a hydrophilic anionic porphyrin induced lipid peroxidation

and inactivation of succinatc dehydrogenase in isolated mitochondria (Chatteqjee et ill.)

1997a). Relaxation of supercoiled plasmid D‘tc:A was also observed (Chatterjee el al.,

199717). It is unclear whether similar effects on mitochondria could be observed when

cells arc incubated with this compound, since localisation in mitochondria is

unexpected.

26 4. Introduction

42.1. Apoptotic cascade

Apoptosis, also termed programmed cell death, is a highly regulated cascade of

events, comprising, (1. s., release of cytochrome c from mitochondria, subsequent

activation of caspases, loss in AY, membrane blebbin,, 0 nuclear condensation and DNA

cleavage. Morphological and biochemical changes during apoptosis lead to cell

degradation, different from necrosis where a decay of the plasma membrane takes place

(Fig. 2). Apoptosis is critical for development and tissue homeostasis, and mutations in

one or several proteins participatiu g in its regulation or execution may lead to

oncogcnesis (Evan and Littlewood, 1998).

cell dehydration

APOPTOSIS-

apoptotic bodies

signal or treatment

LY chromatin condensation @-%Y&

nuclear fragmentation membrane integrity

preserved

cell and mitochondrial plasma membrane

swelling rupture

NECROSIS -

Fig. 2. Apoptotic and necrotic cell death (modified from Darzynkiewicz et ul., 1997). In apoptosis,

n~oqYl~ological chaqes such as cell shiRkage, chinatin condensation, DNA fragmentation, aid

fomution of apoptotic bodies take place. In necrosis. the cell anti initochondria swell and the plasma

membrane ruptures.

The apoptotic pathway c,an be divided into three steps: induction, execution and

degradation. Various stimuli, e. g.. mi radiation, staurosporine or serum deprivation,

27 4. Introduction

can induce apoptosis startin, 0 at different sites in the cells, but ending in a common

pathway that leads to controllecl degradation of the cell (for review, see Wilson, 1998).

4.2.2, Caspases

Caspases are cysteinc proteases with a requirement for cleavage after aspartic

acid and are involved in many apoptotic pathways (for reviews, see Nul’iez et al., 1998;

Thornberry and Lazebnik, 1998). They are expressed as proenzymes, activated by

proteolysis, and contribute to apoptosis through direct disassembly of cellular

structures. It is believed that a proapoptotic signal activates an initiator caspase which,

in turn, activates effector caspases, resulting in cellular disassembly. T~LIS, the initial

signal is amplified in a cascade reaction. Different initiator caspases may mediate

distinct sets of signals that linally elicit the same biochemical and morphological

changes.

4.23. Mitochorldria and apoptosis

Mitochondria play a central role in the execution of many forms of apoptosis and

in the initiation of the apoptotic cascade by releasing caspase activating compounds (for

review, see Green and Reed. 1998). During apoptosis. cytochrome c, a component of

the respiratory chain, is released from mitochondria; this is ilihibited by the anti-

apoptotic protein Bcl-2 and enhanced by the pro-apoptotic protein Bax (see paragraph

4.2.4). Cytosolic cytochrome c combines with Apaf- 1 (apoptotic pl-otease-activating

factor) to activate caspase-9 (LA c’r rtl., 1997). III neuronal cell culture models, caspase-9

resides inside mitochondria and is tramlocated to the nucleus upon addition of

apoptosis-inducing agents (Krajewski et (71.) 1999). Wkn the release of cytochrome c

does not trigger apoptosis, c. g., in cells with mutated caspases, cells may die

necrotically as a result of itqaircd electron transport leadin g to generation of oxygen

radicals and decreased production of ATP. Ho\\-ever, cells are also capable of c/e mx~~

28 4. Introduction

cytochrome c synthesis and recovery when the apoptosis-inducing stimulus, e. s.,

growth factor deprivation, is no longer present (Martinou et al., 1999).

During apoptosis, reactive oxygen species are formed in mitochondria and AY

decreases. Initially, these Lvere thought to be early apoptotic steps (Zamzami et (I/..

1995), but later it became evident that formation of reactive oxygen species is a

c.onsequence of cylochrome c loss rather than a cause for apoptosis (Cai and Jones,

1998).

4.2.4, Bcl-2 protein family

Bcl-2 and Bax are proteins 0 f the same structura 1 family, the former being anti-

and the latter pro-apoptotic. The balance between pro- and anti-apoptotic proteins is

believed to determine Lvhether a ccl1 will undergo apoptosis, since they can form hetero-

and homo-dimers. All members of that family contain at least one of the four conserved

motifs known as Bcl-2 homology (BH) domains essential for dimer-isation (for review,

see Adams and Cory, 1998).

Bcl-2 has been found on the mitochondrial outer membrane, endoplasmic

reticulum and nuclear membrane (Lithgow et nl., 1994). Since Hcl-2 has some apparent

antioxidative effects, it was initially believed to inhibit apoptosis by acting as an

antioxidant (Hockenberry et al., 1993). In a cell-free system Bcl-2 prevents release of

cytochrome c from mitochondria, and thus activation of oaspases (Kluck et al., 1997).

Caspase-3 cleaves Bcl-2, resultin_ e in a positive feedback loop and rilrther caspase

activation (Kirsch et al., 1999).

Bcl-2, Bcl-xi and Bax are capable of forming ion channels in membranes and

may thus regulate their pctmeability (Schetldel el nl., 1997; Minn et crl., 1997;

Antonsson et 01.. 1997). Bcl-2 also prevents J.Y loss induced by various reagents, e. g.,

Ca”+ and hydrogen peroxide, in isolated mitochondria. 111 this in vitro model, Rcl-2

maintained AY’ by enhancing proton efllux in the presence of stimuli inducing AY’ loss

(Shimizu et al., 1998). However, this does not stem to be the only function of Rcl-2,

since it also retards the cell cycle (C)‘Reilly er 01., lCYX) and maintains (:a”-‘- homeostasis

in the endoplasmic reticulum (HI: c!f nl., 1907). Bax. in contrast to Bcl-3, induces

29 4. Introduction

cytochrome c release from mitochondria (Manon et nl., 1997; Jurgensmeier et al., 199s;

Eskes et al., 1998). It has recently been proposed that pro-apoptotic proteins such as

Bax accelerate opening and anti-apoptotic ones such as &l-x,, close the mitochondrial

voltage-dependent anion channel and thus regulate AY and the release of cytochrome c

(Shimizu ef ill., 1999).

4.2.5. PDT and apoptosis

Upon incubation of cells \\,ith various PS and irradiation, apoptotic responses are

observed, e. g., cytochrome c release into the cytosol (Vat-nes el nl., 1999), caspase

activity and poly(ADP-ribose) polymerase cleavage (He et nl., 1998), DNA laddering

and fragmentation of nuclei (Lxto et ~11., 1996). Incubation of cells with a I’S and

irradiation can induce either necrotic or apoptotic cell death, depending on the cell line,

PS and incubation protocol used.

Human prostate carcinoma cells (PC3) and rat mammary carcinoma cells

(MTF7) showed an apoptotic response in contrast to human non-small cell lung

carcinoma (H322a) when incubated with photofrin IT’@ and illuminated (He el nl., 1994).

CV-1 cells incubated with photofrin and irradiated underwent apoptosis when incubated

for 24 h with 1 cLgltn1 but not when incubated for 1 h with 10 pgiml (Dellinger, 1996).

P388 murine leukaemia cells sho\ved DNA laddering 1%.hen porphycene dimer or tin

etiopurpurin were used as PS, bul when dicationic chlorin or motiocationic porphyrin

were used, DNA n’as only cleaved to 50 kb particles (Luo et o/. , 1996).

The subcellular localisatiou of the PS seems to delcrmine whether a cell will

undergo apoptosis or not (Kessel et al., 1997). When mitochondria are the subcellular

target, PDT induces apoptosis (Ball et nl., 199s; Noodt et al., 1996). For lysosomal

targeting contradictory results are reported (Brunk et nl., 1997; Kessel and Luo, 199s).

When the plasma membrane is a target, initial apoptosis can be delayed or stopped,

because factors of the apoptotic cascade are Lost from the cytosol (Kessel and Luo,

1998; Kessel et 01.. 1997).

30 5. Aims oC the study

5. AIMS OF THE STUDY

Mitochondria are critical subcellular targets of PDT. 171 vitro and in vim,

lipophilic I’S, e. g., photofrin’, accumulate in mitochondria, and ~tpon irradiation,

structure and functions of this organelle are affected. L,ipophilic cationic con~pounds,

which are taken uy into niitochondria in response to AY, are a promising class 0fPS for

targeting carcinoma cells with elevated A’Y.

mTHPC is a second-generation PS undergoing clinical trials also at the

University Hospital Zurich. Subcellular effects due to ml’HPC-mediated PDT had not

been studied, but there was c\Tidence from microscopy studies that mTHPC also

accumulates in mitochondria. 111 the first part of this thesis, the following questions were

addressed:

0 Does m?‘FTPC accumulate in mitochondria’?

e Are mitochondrial fkctions impaired upon incubation of isolated

mitochondria n.ith m’THPC and irradiation?

* Are mitochondrial functions also affected when ccl Is are incu~lx~ted with

mlXPC and irradiated?

0 Axe there mechanisms other than damage to mitochondria that could elicit

cell death upon mTHPC-mediated PDT’?

* Which kind of photochemical reactions is mediated by tn’:T?TPC (type I or

type II)?

In the second part of this thesis. cytotosicity and phototosicity of three, charged

and uncharged, PS were compared in a pair of cell lines differing in their Bcl-2 content

and AV’ to answer the questions:

* Al-e cytotoxicity and phototoxicity of a lipophilic cationic PS higher in cells

with higher AY’? Is this correlated nith a higher PS uptake?

0 Can the presence of the antiapoptotic protein EM-2 be used to induce higher

cell kill, since Bcl-2 expression correlated mith higher AY in these cells?

0 Is apoptotic cell death elicited in cells after incubation with the PS and

illutnination? Does cytoctxome c release from mitoctiondria take place?

In collal~orations. other aspects of PDT, t!. h ~7~ skin photosensitivity, were to be

addressed.

31 6. Materials and methods

6. MATERIALS AND METHODS

6.1.1. Chemicals

mTHPC was f?oni Scotia Pharmaceuticals Ltd. (Guildford, LJK), TPPSd from

Porphyrin Products (Logan, UT, USA), and VB-BO from Aldrich (Buchs, Switzerland).

mTHPC and VB-BO were dissolved in ethanol, ‘TPPS4 in water. ALA was obtained

from ASAT AG (Zug, Switzerland), Hyperiforce camp. from Bioforce AG (Roggwil,

Switzerland). L4zE was prepared by C. Richter according to Parish et crl. (199s).

Annexin-V-FLUOS staining kit was from Boehringcr Mannheim (R&rem,

Switzerland), and nitrate/nitrite tluorometric assay kit from Cayman Chemical (Alexis,

L;iufcIIingen, Switzerland). I)il?ydrol’17ociat~liae 133 and flue-3 AM were purchased

kom Molecular Probes Europe (Lciden, Netherlands).

Dulbecco’s modiiied eagle’s medium, Opt&EM, RPM/11 1640, Hank’s buffered

salt solution. foetal calf serunl (FCS)? penicillin, streptomycin, and trypsin/EDTA were

from Gibco (Basel, Switzerland), Ham’s F-l 2 medium without phenol red lkom

I3ioConcept (Basel, Switzerland). G-418, MTT and other chemicals were from Flulta

(Buchs, Switzerland). Mouse tnonoclonal antibody clone 7H8.2C12 recognizing mnurine

cytochrome c was purchased from RDI (Research Diagnostics, Flanders, NJ, USA), and

anti-mouse El-2 rabbit polyclonal antiserum from lipstate Biotechnology (Lake Placid,

NY, USA).

C2 and CG ceramide ~‘ere obtained from Alesis Biochemicals (L.kfel fingen,

Switzerland) and DHC from Calbiochem (Lmern, Switzerland). Cerarnide stock

sol&ions were prepared in ethanol containing 1 0% DMSO. Daylong@ 16 sunscreen and

Excipial” light and dark cm~ercreams were from Spirig AG (Egerltingen, Switzerland).

Other protective substances tested were made at Kantonsapotheke (Ziirich,

Switzerland).

Other chemicals were purchased from standard suppliers (Fluka and Sigma,

Buchs, Switzerland).


6.1.2. Cell lines

The MCF-7 cell line is derived from a human breast cancer and has a doubling

time of 1.3 days. MCF-7 cells lvere grown in OptiMEM supplemented with 20 % FCS,

25 TUhl penicillin, and 25 n&ml streptomycin.

Mouse fibrosarcoid L929 cells were transfected with a vector alone (BV3) or

containing the human 1~1-2 gene (B22; Hermet et al., 1993). They were grown in

TAlbecco’s modified eagle’s medium containing 10 % KS, 25 IU/ml penicillin, 2.5

mg/ml streptomycin, and 0.5 m&l G-418 in every third passage. Overexpression of

B&2 in 1322 cells was reconfirmed by Western blot analysis.

Small cell lung cancer cells (N417) ovcrcxpressing Bcl-2 in different levels

(control, Bcl-2*2, Bcl-2*3) were obtained from Dr. U. Zangemeister-Wittke. They were

grown in RPM1 1640 supplemented with 10 9’0 K’S, 4 mM L-glutamine, 25 IUiml

penicillin, 25 mg:‘ml streptomycin, and 0.8 tng,/ml G-41 S in every second or third

passage.

HaCaT ccl is, a l~u~nan keratinocyte cell line. were grown in RPMT 1640 medium

cnuiched with 10 ‘li;, FCS, 4 mM L-glutamine. 25 IUiml penicillin, and 25 n&ml

streptomycin.


6.2.1. Isolation of mitochondria

Female M;istar rats (200 - 250 g body \veight), which had been fasted over night?

were killed by decapitation. Their livers wcrc quickly removed and washed in 30 n-11 of

ice-cold 210 t&l n~annitol, 70 tnM sucrose, 5 rnM Hepes, 1 mM EDTA, pH 7.35

(buffer A). Fat and connective tisslte were removed. The livers were cut into small

pieces and homogenised in 60 ml of buffer A per liver using a glass homogeniser with a

Teflon pestle. Mitochondria were isolated by differential centrifugation (Schlegel el nl.,

1992) as fbllows: All centrifugation steps were done at 4 “C. The hotnogenale was

centrifuged for 10 min at 1’000 g (2’900 r-pm) in a Sorvall centrifuge model RC-5B

with an SS-34 rotor. The supematant was centrifltged for 10 tnin at 10’000 g (10’000

rpm). Fat bloating on top of the supernatant was removed with Linsoft tissues, and the

supernatant was poured off. The mitochondrial pellet of each liver was resuspended in

80 ml of buf’fer 13 (buffer A without EDTA) using n reagent tube filled with ice. The

suspension was cctitrifitged for 5 tnin at 1’000 g, and the resulting supetnatant was

centrifuged for 10 min at 10’000 g. The pellet was resuspended in buffer B, and

mitochondria from two tubes wet-e combined and pul: into one tube. Mitochondr-ia were

centrifuged again for 10 min at 10’000 g. The final pellet was suspended in buffer B to

<rive a protein cotxentration of SO - 120 tng/tul, Mitochondria were stored on ice. The ‘3

protein concentration was determined by the biurcl method with bovine serum albutllitl

(BSA) as standard (Schlegel ct ul., 1992).

6.2.2. Tncubation of mitochondria with mTHPC and irradiation

Mitochondria wetx suspended in 210 mM tnannitol~ 70 mM SWXOSC, 5 tnM

Hepcs, pH 7.35 at 4 “C (buffer B) or in 780 tnM KCl, 10 tnM trjs-jhydroxymethyl)-

atninotiiethat~c, pF1 7.35 at 4 “C (buffer C) at a concentration of 3 mg of protein/ml.

Portions of 5 ml of suspension wcrc placed in Petri dishes of 60 mm in cliatuetcr.

tvTHPC was added to final concentrations of 0, 5, 15 and SO ~.tg/mg mitochondrial


protein. Mitochondria were incubated for 1 h at 4 “C in the dark. They were

subsequently irradiated under continuous stirring for 5 min at 652 nm with a power of

50 mW (5.3 J/cm’) using a diode laser (Applied Optronics Corp., South Plainfield, NJ).

Samples for protein oxidation and lipid peroxidation measurements were frozen and

stored at -SO “C. All samples were protected from light before and after irradiation.

6.2.3. Determination of protein oxidation and lipid p-oxidation

6.2.3.1. Protein oxidation

Protein oxidation was determined with Nal3”FI~ as described (Levine et rrl.,

1990). Two hundred I.L~ of mitochondrial suspension were incubated for 1 1~ at 37 “C

with NaB’Hd (dissolved in 0.1 M NaOH and stored at -SO ‘C), which reduces carbonyl

groups to alcohols, thereby introducing a stable tr-itium label into the proteins. The

reaction was stopped and the proteins were precipitated by the addition of 1 ml of cold

20 % trichloroacetic acid (w/v), The proteins were separated from the reaction mixture

by filtration through glass microfZ~re filters (Whatman, Maidslone, UK). Unbound

radioactivity was removed by thoroughly washing the filters with 10 ml of 10 %

tr-ichloroacetic acid. Radioactivity remaining on the filters was counted in a p liquid

scintillation counter (Kontron Instruments). Quenchin, * due to mTHPC was corrected

for.

6.2.3.2. Thiobarbituric acid-reactive substances

Thiobarhituric acid-reactive substances (TBARS) ~vet’e measured as described

(Buege and Aust, 1978). Two hundred ~1 of mitochondrial suspension (2 mg/ml) were

mixed with 1 ml of a solution containing 15 $4 trichloroacetic acid, 0.375 O/o

thiobarbituric acid, 0.25 M HCI, 0.24 ITIM FeClj and 50 ;IM butylated hydroxytoluene.

The mixture was heated for 15 min in a boiling n.ater bath, cooled on ice and

ccntrifLlged to remove the precipitate. The absorbance of the sample was determined at

535 111'11 (~cl,lo,n,,,,,,,,-::1.5(ihlo' M’ cm”) against a blank containing all the reagents

folios the mitochondria,


6.2.3.3, Lipid hydroperoxides

Lipid hydroperoxides (LOOH) were determined by the iodometric assay as

described (Buege and Aus?, 1978). One ml of mitochondrial suspension (2 mg/ml) was

mixed with 5 ml chloroi’orm:methanol (2 vol: 1 \-ol), followed by centrifugation at 1’000

g for 5 min to separate the phases. The lower layer (3 ml) was taken to dryness in a 45

‘C water bath under a stream of nitrogen. One ml of acetic acid:chloroform (3:2) and 60

mg potassium iodide (0.05 ml) tvcrc then added. After mixin g, the samples were placed

in the dark for 5 min, followed by addition of 15 mg cadmium acetate (dissolved in 3 ml

of H1O), The solution was mixed and centrifuged at 1’000 g for 10 min. The absorbance

of the upper phase was determined at 353 nm against a blank containing the complete

assay mixture Wrttrs the mitochondria. Unfortunately, the hydrophobic mTHPC was

also extracted by the procedure described above. Since its interference in the following

steps could not be prevented, only samples containing the same amount of mTHPC

were compared.

6.2.4. Determination of mitochondrial oxygen consumption

Mitochondria (2 mg/ml) mere incubated in buffer B under constant stirring at 25

“C in the presence of 1 mM ethylene glycol bis(p-aminoethylether)-N,N,N’,N ‘-

tctraacetic acid (EGTA; 1.3 111) in a closed chamber (1.25 ml) protected from liglzt with

alumil~ium foil. Mitochondrial respiration was measured with a Clark-type oxygen

electrode (Yellow Sprin g Instruments, Yellow Springs, OH), (Estabrook, 1967).

Respiration dependent on complex I was induced by the addition of 2.4 mM K’-

pyruvate and 1.25 m&f Kim-malate (5 111) and inhibitecl by 5 pM rotenone (3.25 pl),

respiration supported from complex II by the addition of 2.6 mM K-‘-succinate (6.5 ill),

and respiration of complex 11~’ by the addition of 0.5 1-19 antimycin A (2.5 111; to block

con~plcs III) and 10 mM ascorbate,~O. 17 mM tetratnethylphcnylenediamine

(Asci’TMPD, 21 .S 1.11). (I, CC er 01.. 1067: Tyler and Gonze, 1967).


62.5. Determination of the mitochondrial membrane potential and of Ca2+ uptake

Mitochondria were incubated at 25 “C with continuous stirring and oxygenation

at a protein content of 1 mg/ml in 2.4 ml ofbufYer B (pH 7.2 at 25 “C). The membrane

potential (AY) was measured in the presence of 10 pM safranine T (Akerman and

Wikstroem, 1976) in an Aminco DW-2a spectrophotollleter at 5 11 - 533 run. The

distance between the highest and the lowest absorption dift‘erence (AAA) was

determined after Ihe addition of 5 PM rotenone,!? .5 mM I<‘-succinate and 0.5 @l

carbollylcyatlide-3-chlorophen~ll~ydrazo~~e (CCCP). respectively.

Ca-?‘. uptake and release n’as measured in the presence of 50 pM 3,6-bis[(2-

a~senophenyl)azo~-4,5-dil~ydroxy-2,7-naphtl~alene-dis~~lfonic acid (arsenazo TII). Ca”’

movements across the inner mitochondrial membrane wet-e monitored in an Aminco

DW-2a spectrophotometer at 675 - 685 nm (Liitscher et nl., 1980; Schlegel et ctl., 1992).

After addition of 5 pM rotenone, mitochondria were energized wit11 2.6 mM f<“-

succinate and loaded with 40 umol Ca2+/mg ofmitochondrial protein.

6.2.6. Extraction of mTHPC from mouse liyer mitochondria

Nude mice were injected i. p. with 0.1, 0.15 or 0.3 rng mTHPC/kg body weight.

After 48 h liver mitochondria were prepared as described above and stored at -80 “C.

Extraction ofmTHPC was performed in collaboration with Dr. W. Blodig (Institute of

Biocheniistry) as described (Whelpton et a/. , 1995) with minor modifications. The

extraction solution consisted of MeOH:DMSO (4: 1) to which I-I;?0 had been added at a

ratio of MeOH/DMSO:H$) (40: I). T\vo hundred 1.11 of mouse liver mitochondria were

mixed with 400 pl of extraction solution and shaken. They were centrifuged at 14’000

i-pm in an Eppendorf centrifuge for 2 min. Four hundred !11 of the supernatant were

mixed with 200 111 H~O. 7’~vo hundred ~11 \~ere irl.jected into the HPLC. JTI'IXPC was

detected by absorbance at 423 ntn (~efcrcnce 510 * 10 rm). The mobile phase was

acetonitrile:O. 1% trifluoroacctic acid (77:23).


6.2.7. Confocal laser scanning microscopy

For confocal microscopy stlldies a Leica ‘KS 41) microscope (Leica,

Glattbrugg, Switzerland) cquippcd Lvith an ArKr laser was used.

For determination of subcellular localisation cells were grown on coverslips and

incubated for 1 h with 1 PM VR-HO, for 24 h with 1 PM mTHPC or with 10 HIM

TPPS.,, or as indicated in the Figures. Dyes \~‘ere exited at 568 nm and fluorescence

above 590 nm was detected lvith a longpass filter,

When cells were doubly stained with

diliydrorl~odamiIle 123 (DHR) and mTHPC,

excitation was at 488 nm, RI1123 fluorescence

was detected with a PITC bandpass filter and

n$TFIPC fluorescence above 500 nm with a

longpass filter, There was no cross-talk

between the dyes as coniirmed by deter-

mination of dye 0uorescence in a iluorimeter

(Fig. 3).

For dctcction of propidium iodide (PI)

and annexin V (,4V) binding, cells were grown

on 8 chamber microscope slides, incubated

with 1 PM VB-HO for 1 h and illuminated for

6 min, with 10 pM TPPSJ Car 24 h and

illuminated for 2 min or with 0.1 1tM mTHPC

for 24 h and illuminated for 1 min.

Immediately or 30 min after illumination, cells

were rinsed with HBSS and stained with (IV

and PI for 15 min as described by the

manufacturer of the kit. Excitation was at 4SS

I / !L; + Rh123

“i -.-...-

500 600 700 nm

mTHPC

:i, I

, Ji e---..-_.. ---l -_ / I /

500 600 fl

700 nm

/ I I / ,

500 600 700 nrn Fig. 3. Fluorescence emission of Rli123

nm, emission of AV was detected with a FITC bandpass filter, emission of PI with a

590 11117 lollgp”s” filter,

For estimating intracellular Calf concentrations, cells were incubated with 4 HIM

flue-3 AM for 30 min at 37 “C. Fluorescence was excited at 488 nm and detected with a

FITC bandpass filter.


6.2.8, Fluorescence microscopy

For uptake studies, fluorescet~ce of the dyes was detected using a Lcitz DMRBE

microscope (Lcica, Glattbntgg, Switzerland) equipped with a col~lputel--co1ltrolled

charge coupled device camera (Photometrics Ltd., Tucson, AZ, USA). For excitation a

.530/595 nm bandpass filter, and for detection of emission a 615 nm longpass filter were

used. Five micrographs per slide were taken, and 2 cells per picture were analysed.

Experiments were repeated 5 times. Analysis of the pictures was done with TPLab

Spectrum software, version 3. la (Signal Analytics. Vienna, \‘,4, USA). IlJptalte of

positively charged dyes into cells can be determined by fluorescence microscopy and is

a measurement of relative mitochondrial membrane potential (Johnson ef nl., 1980).

62.9. Irradiation of cells with laser light

Cells were irradiated using a diode laser (14pplied Qtronics Corp., South

Plainfield, NJ, U. S. A.) ivhich emitted light of 652 nm and was connected to a ffont-

lens light diffrsor FIX (Medlight SA, Lausanne, Stvitzerland). The power being set to

25 mW, irradiation times of 15, 30, and 60 s corresponded to energy closes of 0.13, 0.26,

and 0.53 Jicm2, respectively. Power was measured with a Fieldmaster powermeter

(Coherent Inc., Santa Clara, USA).

62.10. Illumination of cells with white light

Cells were illuminated with an Intralus’ ?vX)R 1.00 lamp (Volpi AC, Schlicren,

Switzerland) containin g a 100 W xenon lamp emitting at 400 - 780 nm. The light

intensity was 4.1 x 10’ lus correspondin g to a power of 60.4 ndWcm” at 55s nm

according to the manufacturer‘s manual.


6.2.11. Colony fortnation assq

Per Petri dish 200 (MU-7) or 300 (HaCaT) cells were seeded. and after 24 h the

PS (mTHPC or as indicated) \vas added. After 24 h (efl’ects of antioxidants) or 48 h (CF

vs. oxygen consumption) incubation time the cells were irradiated. Subsequently, the

medium was renewed, and cells were incubated for 7 or 8 days. They were then fixed

with MeOIliacetic acid (3: 1) and stained with Gemsa solution, and colonies (X0 cells)

were counted,

62.12. MTT assay

Adherent cells (R22> RV3) were seeded into 06 well plates 24 It before addition

of the PS in a volume of 200 111. After incubation in the dark or 24 h after illumination,

medium was replaced by fresh medium containing 0.5 mg MIT/III 1, and cells were

incubated for 3 h at 37 “C (Momann, 1983). Medium was replaced by DMSO and

absorbance was read in an ELBA reader at 540 nm (Twentyman and Luscornbe, 1987).

The surviving fraction was calculated using a standard curve of untreated cells present

on the same ELEA plate, Each experiment was repeated 3 to 5 times. Duplicates

(illuminated cells) or tt-iplicatcs of each experiment (standard curve, cells incubated in

the dark) were obtained.

Suspension cells (N317) \vere seeded into 06 n,ell plates 24 h before addition of

the PS in a volume of 100 L11. *After incubation in the da-k or 2-1 12 after illumination, 10

111 of NT’I’T (5 mg’ml, final concentration 0.45 mg?nl) were added and cells were

incubated for 3 11 at 37 “C. One hundred ~1 lysin g solution (250 in1 water, 250 ml

dimetllylfor~~~amide, pH 4.7 adjusted with 80 % acetic acid!20 % 1 M HCl, IO0 g SIX)

was added. Lysis of cells took place at 37 “C overnight and absorbance was determined

at 540 ml.


6.2.13. Determination of cellular oxygen consumption

About 10’ MCF-7 cells ~vere seeded per culture flask. Incubation with mTHPC

and irradiation was performed as described above. After irradiation, the cells were

trypsinised and counted. Oxygen consumption of 3x10” cells in their pyruvate-

containing medium was measured with a Clark-type oxygen electrode at 37 *C under

continuous stirring.

62.14. Western blot analysis

For the detection of cytochrome c release, cells grown in Petri dishes (10 cm

diameter) were incubated with 1 pM VR-BO for 1 h, 0.1 bll\/I mTFIPC or 10 HIM TPPS,,

for 24 11, and subsequently illuminated for 8, 2 or 5 niin, respectively. One 11 later cells

were trypsinized, and s~hzell~~lar- fractions were obtained as described (Liu et nl., 1996)

with minor modifications. Cells were resuspended in 200 111 of an extraction solution

(20 mM Km’-Hepes, pH 7.5, IO n&I KCI, 1.5 mM Mg$X, 1 mM I(‘--EDTA> 1 mM Na’-

EGTA, 1 mM dithiothreitol, 250 mM sucrose, 0.1 mM PMSF, 5 yglml pepstatin and 2

~1gimI aprotinin). After sitting on ice for 15 min. they were disrupted by 20 strokes of a

syringe through a 25 G needle. Suspensions were centrifuged in an Eppendorf

cetitrifLige at 1000 g for 10 min at 4 “C. The pellet contained the nucleic fi’action and

was resuspended in the extraction solution. The supernatant was centrifkgcd at 10’000 g

for 1S min. ‘The resulting pellet contained the mitochondria and was resuspended in the

extraction solution. The supernatant \5‘as centrifuged it1 a Beckman TL-100

ultracentrifuge at 100’000 g for 30 min. The pellet was discarded, the supernatant

contained the cytosolic proteins. All samples were stored at -80 “C. The following steps

were performed by S, Mocha (Department of Radiation Oncology, University Hospital

Ziirich): Proteins from cytosolic fractions (150 btg) were precipitated with

trichloroacetic acid, resolved by SDS-polyacrylamide gel electropboresis (15 O/; SDS)

and blotted onto PVDF membranes. Antibody clctec,tion was achieved by ECL,-

enhanced cher~~ilu~l~inescellce (Amersham, Diibendorf, Switzerland) using a horseradid~

peroxidase-conjugated secondary antibody, according to the manufacturer’s protocol.


62.15. Nitrate and nitrite determination

For the detection of nitrate and nitrite in the supernatant of the cells, 5000 MCF-

7 cells per well were seeded in a 96 1~11 plate. After 24 11 the OptiMEM was replaced

by Ham’s F-12 medium. and 0.1 1-19 mTHPC/ml was added. Ham’s F-12 medium

contains neither phenol red nor nitrate and nitrite that would disturb the assay. After 24

11 incutl7ation time, cells were washed with HRSS, then kept in 40 yl of Ham’s F-12

medium, and irradiated during 30 s with 25 mW (0.26 .T!cm”) laser light of 652 nm

wavelength. At the indicated times, 30 ~1 of the supernatant were take11 and stored at

-20 “C until analysis.

For the detection of nitrate and nitrite in the cytoplasm of the cells, 250’000

MCF-7 cells were seeded per Petri dish (diameter 6 cm). After 24 h 0.1 1~8 n1’T’f-TPC/~nl

was added. After 24 11 incubation time, cells were irradiated during 30 s with 25 tnW

(0.26 J/cm”) lam light of 652 ml wavelength, ,4t the indicated times, the cells were

rinsed with cold Ham’s F- 12, scraped off, and centuifnged. The supetnatant was

discarded, and 50 ~1 of a lysis solution containing 0.1 M NaCI, 0.01 M ‘Tris, 1 rnM

EDNA, 4 ‘3/o SDS, 1 pg/ml aprotinin, 100 kLg/ml PMSF, and 1 ~~g/nll pepstatin, was

added, The cells were resuspended, vortexed, and cents-iftrged. ‘Tie supernatant was kept

at -20 “C until analysis.

“l’wenty 111 of the supernatant or 10 pl of the cell extract were incubated with

nitrate reductase and enzyme co-factors for 2 II according to the protocol of the kit, 2,3-

dia~ninol~~~hthaler~e was added, and after 10 min the reaction wvcs stopped by the

addition of NaOH. Fluorescence was excited at 365 nm, and emission at 405 or 408 nm

was recorded (,samples from supernatant or cell extract. respectively).

6.2.16. Statistical analysis

Statistical analysis of experiments \vith isolated mitochondria or MCF-7 cells

(CF, oxygen c~onsumption) ~vas performed usins a Student’s t-test. Data are expressed

as means i standard errors (SE) of 3 to 4 experiments. Statistical significance is

indicated as: *, 0.05 > P > 0.01: **, 0.01 > P > 0.001; ***, 0.001 r P.


Statistical analysis of experiments with B22 and BV3 cells (dye uptake,

survival) was done usin g a Wilcoxon-Mann-Whitney test, a = 0.05. Data are expressed

as means % SE of 3 to 5 experiments.

43 7. Kesults

7. RESULTS

7.1. Pl~otoserzsitisntioIt of isolated nzitocltortdrin by m THPC

7.1.1. Protein oxidation and lipid peroxidation

Oxidative ntodi fications in proteins and lipids of isolated rat liver mitochondria

incubated with increasing concentrations of mTHPC and irradiated with laser light were

assessed by measuring the amount of carbonyl groups and TBARS, respectively.

Neither incubation with tnTHPC nor irradiation alone affected these parameters, but

when applied in combination an increase in carbonyl groups (Fig. 4) as well as TBARS

(Fig. 5) was found. The extent of oxidative modifications depended on the

c,oncentration of mTHPC present.

0 5 15 so

,ug rnTHPC/rng protein

44 7. Results

4 7 nnot irradiated

0 5 1s SO r-18 mTHPC/mg protein

Fig. 5. Formation of ‘I’HARS in rat liver mitochondria incubated in buffer 13 with increasing mTFTPC *

Iii order to investigate the mechanism - type I ot’ type II - of m’TWPC-mediatecl

phototoxicity, the effects of antioxidants as well as I)?0 on the fomation of LOOH,

TBARS and carbonyl groups lvere determined (‘Table 4). The hydroxyl radical trap

mannitol had no effect on the oxidation of proteins and lipids, possibly because the

reactive oxygen species were produced inside the metnbl-anes? to which nlannitol has no

access. In contrast, the lipophilic antioxidant butylated hydroxytoluene (BFIT)

decreased the amount of all oxidation products investigated. Preparing the buffer \vith

D70 instead of El:0 clearly, although statistically not significantly (due to the

limitations of this assay as mentioned in Materials and methods), stimulated the

formation of LOOH. Finally, the singlet oxygen scavenger dimethylf~~ran (DMF)

diminished the amount of LOOH. Taken tog&her, these results indicate a mechanism

composed of type T as lvell as type II reactions.

45 7. Results

Table 4. Effects of inhibitors of reactive oxygen species and effect of D20 on the formation of lipid

hydropcroxides (l,OOH)n, thiobarbituric reactive substances (TBARS)” and carbonyl groupsb induced by

incubation of rnitochondria in buffer C with 15 11s n~TIIPC/mg protein for 1 h and irradiation with 5.3

J/cm’. As indicated. 100 mM mannitol, 100 1A4 bcltylated hydroxytoluene @FIT) or 100 I-IN

dimethylfuran (D&W) n;e~ prcsc’nt during the irradiation. 01 the incubation buffer (buffer C) was prepared

using I&O.

LOOH

O/o of control i SE

-WARS

% 0 f control f SE

carbonyl groups

16 0 f control 3: SE

control 100.0 rk 45.3 100.0 It- 1 1.7 100.0 3: 5.2

100 mT\il nmnitol 101.7 :k 35.9 101.0 i 16.2 105.0 $I 11.0

100 pM HT-u 7S.G i 35.7 35.8 -i- 2.4 72.9 rt 5.3

in D20 118.5 i 56.3 91.3 + 9.8 90.1 * 7.5

100 Ql DMF 75.0 i 16.5 103.3 * 13.3 113.5 f 9.9


Table 5 shows the effects on respiration of incubation with different

concentrations of mTHPC and irradiation. Oxygen consumption stimulated by 2.4 mM

I<‘--pyruvateil .35 mM I<-‘--malate ~ic1 comples 1 was increased at the lowest m’IXPC

concentration used, but decreased at higher concentrations, Oxygen consumption

stimulated by 2.6 mM T&uccinate via complex II and 10 mM ascorbate/0.17 mM

tetrallietliylphellylet?edialllille ~+a complex 11; decreased with raising mTHPC

concentrations; complex II \vas more sensitive than complex IV. Addition of 0.8 l&f

CCCP, an uncoupler of tnitochondria, showed little effect on oxygen consumption or

mitochondria that had received I~THPC plzrs it-radiation, obviously because they were

already uncoupled. The ratio of the rate of oxygen consumption during (state 3) and

after (state 4) ADP-stimulated respiration (respiratory control index) was also

determined for complex Il. Mitochondria n’erc incubated in the presence of 1 n&I

KH;?POJ. Addition of 75 nmol ADP did not result in an increased oxygen consumption

in any of the pllotodynamically treated samples (Fig. 6, only the sample with lowest

mTHPC concentration sho~vn),

46 7. Results

Table 5, Oxygen consumption supported from different respiratory complexes of mitochondria incubated

with n~‘l’tlPC and irmdiatcd \vith laser light (5.3 J/cm”). For experimental details see Materials and

Methods. ~4. Statistically significant differences (not irradiated w. irradiated): *, 0.05 2 1’ > 0.01; *“i,

0.01 2 P z 0.001: ***, 0.001 2 1’.

Complex I

Co1nplex II

Cornp lex IV

“”

..“I,

.I-^

5 103.0 :k 11.3

5 86.3 h 7.7

15 100.4 :L- 11.2

50 94.6 i 7.8 “., . .-,_ .--_.-^--“.x.“.._ ..,.. - .,.,..... _ _“-.---.-.. _... ..~.” “x_” “. ““. “l

0 100.0 * 5.1

s 99.2 :rtz 7.1

IS 94.3 -i 5.9

50 95.2 I 4.5

102.5 :k 2.5

94.9 :k 7.4

9 1. I :!: 4.7 53.3 + 7.7 **

Fig. 6. ADP-stimulated oxygen consumption in rat liver mirochondria. Mitochondria were incubated in

b~lffer 13 at 25 “C in the presence of 1 1~51 EGTA. 5 1111 rotenone, 2.6 I&I K’.-succinate. and I nM

KH2PO+ At the solid arrows, 75 nmol .4DP XYIS added. and at the dashed arrows, 0.8 @I CCCP was

added, (a) LJntrcated tuitochondria. (b) \litochondria irradiated during 5 min with 50 mW of light of 652

IUII (5.3 J/cm’). (c) Mitocl~ondria incubated with 5 ;lp m’TTIPC/mg protein for 1 Ii in the dark. (d)

Mitochondria incubated as in (c) and irradiated as in (b). One espcriment typical of four is shown.

47 7. Results

7.13. Decreased mitochondrial membrane potential and Ca2+ uptake

Since ATP production in mitochondria depends on AY?, we were interested in

this parmeter of n~embrane intactness. Fig. 7A and B show how ALI’ is affected by

PUT: irradiation in the presence of as little as 5 ~[g m’TFfPC/mg protein diminished the

distance between the higllest and the lowest absorption difference by about half, and

irradiation in the presence of 15 pg tnTHPC or moreimg protein completely prohibited

build-up of AY. Roth the rate and the total amount of Ca”” taken up by mitochondria

incubated with 5 1-1 g mTHPC/mg protein and irradiated were clearly diminished as

compared to controls (Fig. 8).

rotenone

AA+l.O 1

I

1 min - CCCP

48 7. Results

+ not irradiated

Fig. 7. Changes in the membrane potential of rat liver rnitochondria incubated wit-11 mTHPC and

irradiated with laser light. (A, a) Untreated mitochondria. (b) Mitochondria incubated with 5 lrg

1n’I’1-1PC/r~~g protein for 1 11 and irradiated during 5 min with 50 mW of’ light of 652 nm (5.3 J/cm”). For

details see Materials and methods. One experiment t)rpical of four is shown. (U) Distance between the

highest and the lmvest absorption difference (&!A) after the addition of rotenone/K’-succillatc and

CCCP, respectively. \‘alues are means i SE from three ( 15 an 50 ~1s m’I’HPC/mg protein) or fo~n (0 and

5 1.18 unTHPC/mg protein) csperiments.

t 1 rotenone ca’- rotenone ca”‘-

.\n=o.os ! )

2 Illill

L (

\

succinate succinate

1

c ,J”L

t t d -

t t rotenone (y. rotenone ca-: 1.

Fig. 8, Calm’- uptake b>~ tnitoclmndria incuhat?d nit11 n’~l’HPC and irradiated with laser light. At the arrows,

5 1tM rotenone. 2.6 n&I K~ -succinatc, and 40 nmol Ca’.- ng 01‘ mitochondrial protein was added. (a)

Untreated mitochondria. (b) Mitochondria irradiated during 5 niin I\-ith SO mW of light of 652 ml (5.3

J/cm”). (c) Mitochondria incubated \yitli 5 11s mTHPC~mg protein for 1 11 in the dark. (d) Mitochondria

incubated as in (c) and irradiated as in (b). One experiment typical of three is shown.

49 7. Results

7.1.4. Accumulation of m’I’HPC in mouse liver mitochondria

To answer the question \vhether mTHPC would also GI vim accumulate in liver

mitochondria. nude mice \verc injected i. I-,. n-ill> 0.15 or 0.3 mg mTHPC/kg body

weight 4S h before preparation of liver mitochondria. IIITHPC conteds weye

determined by HPLC (Fig, 0). Retention time of pure mTHPC was 7.3 min, and of

m’I’HPC extracted from mitochondria 8.0 mill under these conditions. The peak shortly

before 3 mill in Fig. 5% may be a degradation product ofm”THPC, since it is not present

in pure mT~HPC (a) or when mTHPC was added to isolated nnitochondria inmcdiately

before extraction (d).

a

mAV

c

mAV li 84

1 j

4; ? i

0*

time (min)

j i mTHPC3

b

16

12 mAV

8

\ *-\-\r,~.- 0

2 4 6 8 10 12 time (min)

time (min)

i T \ ,,“.

, Y-i

2 4 6 8 IO time (min)

50 7. Results

In another set of experiments, mitochondria from 5 mice injected with 0, 0.15 or

0.3 mg mTHPC/kg body weight 4S 11 before isolation of mitochondria were obtained

and analysed (Table 6). Unfortunately. the yield from the extraction procedure was low,

and the HPLC peaks were too small to be quantitatively analysed. Therefore, this study

was not continued.

Table 6. Estraction ofmTHPC from mouse liver mitochondrin. Mice were injected with 0, 0.1 S or 0.3 mg

mTIIPC/kg body might 48 11 before isolation of mitochondria. For details of extraction procedure, see

Materials and methods.

mouse mg mTHPC/kg body calculated amount of mTHPC in liver mitochondria weight (ng mTHPC/mg mitochondrial protein)

0

0.15

0.15

0.3

0.3

(peak area too small)

(peak area too small)

0.9

0.55

51 7. Results

7.2. S~chcellular events after photosensitisation qf MCF-7 cells by

m THPC crnd irradiation

7.2.1. Subcellular localisstioll of mTHPC

Incubation of MCF-7 cells for 6 11 with mTFIPC gave weak fluorescence in the

cytoplasm (Fis. lOa) as determined by con focal laser scanning microscopy (CLSM).

After 24 11 the fluorescence became brighter and the nuclear menhrane was distinctly

stained (Fig. 1 Ob). After incubation for 48 and 72 11 membranous structures iu the

cytoplasm were more clearly seen (Fig. 1 Oc and d). No fluorescence signals were

\ _, ,\, \\ \~ _\\ \,, _ __ \ \\,\~\ > \\ ~ \\ ._~ \\\ \~ \_I \

Fig. 10. m’EIPC fluorescence in MCF-7 cells. MCF-7 cells rvere incubated with 1 pg tnTHPC/ml for 6 II

(a; 64 pm x 64 [ml), 24 h (b; 64 bun x 64 pm), 48 h (c; 62 11m x 62 ~~111) or 72 11 (d: 100 ptn x 100 pm).

Fluorescence was detected by CULL excitation was at 488 mn, emission was detected with a 590 nm

longpass filter.

52 7. Results

detected within the nucleus at any time during these experiments.

To investigate the possible localisation of mTHPC in mitochondria, MCF-7 cells

were also stained with dihydrorhodamine 123 (DHR). The fluorescence patterns of

rhodalnine 133 (Rh123), the oxidation product of DHR, and m’THPC were not identical,

RI1 123 localised aronnd the nucleus (Fi g 1 1 a), but m’I’HPC also in the outer regions oi

the cells (Fig. I lb). Some of the structltres stained with mTHPC were identified as

mitochondria by simultaneous staining \\-i th Rh 123 (Fig. 11 c).

In sperm cells m’l’HPC accumulated mainly in the midpiece where the

mitochondria are located, Weak fluorescence S,XS also detected in the cytoplasm of both

head and tail (Fig. 12a and b).

53 7. Results

7.2.2. Colony formation

Tnculmtion with ni’IXPC and irradiation with laser light diminished the CF 01

MCF-7 cells (Table 7). It decreased with increasing tn”IXPC concentrations and energy

doses.

Table 7, Effect of incubation with mTHPC and irradiation with laser light 0x1 CF of MCI;-7 cells, Data are

esptascd in %I of the untreated cont~~fl. II = 3. Statistically significant diffeerences (vs. control): ***, O.iJ()j

2 P.

lllTI-IPc (pg!1nl) irradiation dose (J!cm’) CF * SE significance

0

0

0

0

0.1

0.1

0. I

0.1

0.25

0.35

0.25

0.25

0

0.13

0.26

0.5;

0

0.13

0.26

0.53

0

0.13

0.26

0.53

100.0

97.2

96.7

94.1

94.0

91.7

64.5

6.2

?7.4

34.9

0.S

0. I

54 7. Results

7.23. Decreased oxygen consumption

After incubation with 0.1 keg mT??PChl and irradiation during 30 s, the oxygen

consumption of‘ MCF-7 cells was decreased to 79 % in the rlormal medium (Fig. 13a),

and to 92 %I after addition of c~~~l~onylcq~anide-4-trifluorol~~etl~oxypl~enyll~ydrazo~~e

(FCCP; Fig. 135). incubation with 0.25 pg mTHPC/mI and irradiation with 0.26 J/cm2

resulted in a reduction of osygen consumption to 69 56 and 72 Oh, respectively.

120 100

80 60 40 20

0 i 0

“=?-

t

55 7. Results

7.2.4. Decreased mitochondrial transmembrane potential

To determine whether also AY was affected by incubation of MCF-7 cells with

mTHPC and irradiation with laser light, cells were incubated with R11123, that is taken

LIP by mitochondria in response to AY, before or after irradiation.

When cells were incubated with 0.1 pg mTHPC/ml for 48 h and subsequently

irradiated during 30 s at 25 mW (0.26 J/cm’), a diminished Rh123 fluorescence was

seen after 6 h (not shown), W%en cells were incubated with 1 pg m’I‘fIPC/ml for 68 h

and irradiated during 4 min at 25 mW (2.1 S/cm’), some cells clearly took up less Rh 123

already 1 h after irradiation (Fig. 14a). Those cells and their nuclei appeared swollen

and the plasma membranes disrupted, resembling rapid necrotic cell death (Fig. 14b).

56 7. Results

7.25 Effects of antioxidants

Addition of 100 mM mannitol, 100 MINI butylated hydrorytoluene (BHT) or 100

;LM dimethyl furan (UMF) to the cells 30 min before irradiation slightly increased the

CF as compared to the cells to which no antioxidant had been added (Fig. 15). Refcn-ing

to the antioxidant added, the CF decreased in the following order: mannitol > DMF 1

BHT.

none mannitol BHT DMF antioxidant added

57 7. Results

7.2.6. Increased intracellular calcium levels and nitric oxide formation

mTHPC also localises in the cellular nmnbrane, and the site of PS localisation

determines the site of damage upon irradiation. It was investigated whether upon

incubation with mTWPC md irradiation with laser light the cytoplasmic Ca”.

concentration is elevated as a comequence of membrane damage as it had been reported

for other PS and cells. MCF-7 cells were incubated with 0.1 clg mTFTPC/ml for 24 h and

irradiated with 0.26 J/cm”. They were incubated at various times after irradiation with

flue-3 AM, a cell pcnueable probe that fluoresces nhen Ca’-’ is boulld. Thirty rnin and 1

Fig. 16. lncrcase in intracellular Ca” concentrations of l\ICF-7 cells after incubation with mTHPC and

irradiation with laser light. MCF-7 cells were incubated \vith 0.1 pg mTHlYXn1 for 24 11, and irradiated

during 30 s with 25 mW light of 651 nm (0.26 J/cm’). For half an horn befoCc7rc CLSM pictures were taken,

cells were incubated with t‘luo-.I I\\‘l. (a) Control cells. (bi cells 30 min after irradiation. (c) cells 1 h after

irradation. (d) cells 2 11 after imdation. Picture size is 150 pm x 250 pm. Typical pictures from three

experiments are shown.

58 7. Results

h after irradiation a slight increase in intracellular Ca2’ is detectable as a punctuate

staining (Fig. 16b and c). Two h after irradiation CL? is elevated in the whole cells,

especially in the nuclei (Fig. 16d).

The activity of constitutive nitric oxide synthase (NW) is C;1?-‘--depe*ldcl7t.

Therefore, it was investigated, whether an increase in cytoplasmic Ca2’ concentration

was followed by nitric oxide (NO) formation. After incubation with mTHPC and

irradiation of MCF-7 cells, nitrate and nitrite concentrations in the supematant and

cytoplasm were determined (Fig. 17).

Probably due to the presence of quenching substances in the samples

(supernatant or cytoplasm), their fluorescence was always lower than that of the

standard curve. Therefore. nitrate and nitrite concentrations could only he qualitatively

determined.

As it can be seen iti Fig. 17, the increase in nitrate and nitrite concentrations in

the cytoplasm precccded the increase in the supernatant. Interestingly, there are tlvo

phases of nitrate and nitrite formation. Whereas one phase could be attributed to the

increase in intracellular Cal”, the other one might reflect iNOS expression.

59 7. Results

7.3. Protection against and sensitisation to in vitro photodynamic

therapy by ovewqwession qf Bcl-2

73.1. Subcellular localisation of VB-BO, mTHPC and ‘I’PP& in BV3 and B22

cells

Firstly, the localisation of the three PS in BV3 and B22 cells was analysed. The

positively charged VB-BO accumulated in mitochondria (Fig. ‘1 Sa and d), the neutral,

lipophilic mTHPC was detected in membranous str-ucturcs (Fig, 18b and e), and the

negatively charged TPPS4 was localised in organelles resembling lysosornes (Fig. 18~

and 1) in accordance with previous findings (~~odica-Na~~olitallo (1~ al., 1990; Hornung

et nl., 1997; Malik et al., 1997). Localisation patterns were identical iu both cell lines.

60 7. Results

7.3.2. Uptake of VB-BO, mTHPC and TPPS4 into BV3 and B22 cells

LJptakc of rnTHPC and TPPS~ into BV3 and B22 cells, as quantitatively measured by

fluorescence intensity, 1Yas equal (Table 8). B22 cells, I~owevcr, took up 18 % more

VB-BO as compared to BV3 cells after only 15 niin incubation with 1 ph/l V&B0

(Table 8). VB-BC) uptake depended on AY since it was reduced to a great extent in the

presence of25 p g/n11 nntimycin A and 1.25 phi FCCP (not shown). In all experiments,

muc1~1 shorter incubation times were chosen for VB-BO (15 tnin to 4 11) than for mTT-IPC

and TPP& (24 h), because VB-I30 is taken up rapidly in response to AY’, and not via

diffusion or endocytosis.

VB-HO mTHPC

7.3.3. Dark toxicity and light-induced toxicity of \‘B-RO, rnTHPC and TPP& in

BV3 and B22 cells

Cells were incubated \vith various concentrations of the thee PS in the dark.

Incubation of the cells with the highest non-tosic concentrations was then followed by

illutnillation to elicit phototoxic r’eactions.

61 7. Results

1 x-07 1.-E-06 1 &OS

VB-BO concentration (34)

0 24 48 72 time (11)

00 0 2 4 6 8

illumination time (min)

Fig. 19. Toxicity of VB-RO in T322 and BV3 cells. Cells DYTC incubated with the indicated cottcettbations

(a) or 1 pM VU-T30 (b) for 4 11 in the dark, or n,ith 1 p;CT J”B-HO for 1 h followed by illutnitmtiott (c).

Tlluntination times of 2) 4, 6, and 8 mitt correspotld to doses of 7.25> 14.5, 21.75 and 29 J/cJI?, respecti.-

vcly. Cell survival was determittcd by MI‘T assay (a, c) or by counting the cells up to 3 days ai‘ter tLeat-

ment (h). RV3 cells incuhatcd with VB-BO, II# B32 cells incubated with VB-BO, 0 BV3 cells without

VB-BO, 17 1322 cells without VT3-RC>. * statistically significant difference (BV3 vs. B22, a=O.OS). 11 = 4.

62 ‘7. Results

Imxibation of the cells for 4 h in the dark with 10 pM or 30 pM VB-BO resulted

in higher toxicity in B22 cells than in BV3 cells (Fig. 19a). Since VI%BO targets

mitochondria and the MTT cell survival assay is based on mitochondrial dehydrogenase

activity, a proliferation assay was used to confirm this finding. When incubated even

only with 1 pM VR-I30 for 4 11, a decrease in proliferation of B22 cells as compared to

BV3 cells was found after 45 and 72 h (Fig. 19b). At this concentration of the PS, no

difference in s~mival between the two cell lines was found after 4 h with the MTT

assay. B22 cells also showed higher sensitivity when incubation with 1 pM of the dye

was followed by illumination with white light of400 to 780 11111 (Fig. 1 SC).

Fig. 20 shows dark toxicity (Fig. 20a) and light-induced toxicity (Fig. 20b) of

mTFIPC. No statistically significant differences between BV3 and B22 cells were

found. TPP& was not toxic up to 0.3 n&l and was equally toxic to both cell lines at 1

mM in the dark (Fi g. 21a). The surviving fraction of BV3 cells was lower than that of

B22 cells when incubation with TPPS4 was followed by illumination (Fig. 211~).

1 .E-07 1 .E-06 1 x-05 l.E-04

n~‘111PC concenti~~tion (M)

I 1 n 0.5 1 1.5 2

illwnina tion time (mill)

Fig. 20. Toxicity ofm’EIPC in B22 and PA’3 cells. Cells XYCK incubated with the indicated concentrations

of mTHPC for 24 11 in the dark (a) 01’ mitll 0.1 pM rnTHPC fi,llowed by illwnination (h), and cell survival

was determined by M’l‘T assay (0 BV3 cells, IN B71 cells). Illumination times of 0.25, 0.5, I and 2

tnin correspond to doses of0.91. 1.S 1. .?.62 and 7.25 .l ‘cm’. respectively. I? = 3.

63 7. Results

00 I&06 l.E-0% l.E-0-l l.E-03 0 1 2 3

‘WPS., concentl~~tion (>I) illunha tion time (min)

Fig, 21, Toxicity of ‘TPP& in B22 and BV3 cells. Cells \vere incubated with the indicated concentrations

of TPPS., for 24 11 in the dark (a) or \vith 10 pM 7’P1’S.I follo\ved by illun~ination (b), and cell surviwl

was determined by MT7 assay. Illumination tilnes of 0.5, 1. 2 and 3 min correspond to doses of’ l.Sl,

3.62, 7.25 and 10.87 .T,‘cm’, respectively. @ BV3 cells, I B22 cells, * statistically significant difference

(Bv3 INS. B22, a-0.05). II = 4

7.3.4. Cytochrorne c release, membrane blebbing and annexin V binding in 13V3

and B22 cells

It was also investigated whether phototoxic reactions would induce apoptosis.

Fig. 22 shows cytochrome c release into the cytosol 1 11 after incubation of the cells with

PS and illumination. Cytochrorne c release in B22 was very pronounced after incubation

with VB-BO and illumination as compared to BV3 cells in accordance with the higher

light-induced toxicity of VB-BO in B22 than in BV3 cells. In contrast, incubation with

n~‘Z‘flPC and illumination induced more cytochrome c release in BV3 cells. Despite

higher survival, cytochrome c release in B22 cells after incubation with TITS4 and

illumination was slightly increased compared to BV3 cells.

An early change during apoptosis is the relocalisation of pllosphatidylserine

from the inner to the outer surface of the cell tnembrane. Therefore, binding of AV to

phosphatidylserine on the cell nmnbrane in the absence of nuclear staining with PI is a

marker for early apoptosis. Late apoptosis cannot be distinguished from necrosis by this

method. After incubation \vith the PS and illumination. cells were stained with AV and

PI. BV3 and H22 shelved similar reactions, and examples of both cell lines are shown.

64 7. Results

control VB-BO mTHPC TPPSd

cyto c BV3 E322 BV3 B22 BV3 B22 BV3 B22

Fig. 22, Cytocltronte c release 1 It after incubation of cells with PS and illumination. BV3 and B22 cells

WCI‘C incubated with 1 @4 VB-BO for 1 lt and illuntinated for 8 ruin (V&130), with 0.1 LLM mTIIPC ih~

24 lt and illun~inatcd Soor 2 min (ntTIIPC), or xitlt 10 1&\2 TN’S4 for 24 It and illuminated foor 5 min

(‘I’fY&). Control cells xerc neither inc&ated with PS Itox illmttinated. Cytoso\ic fractions wese obtained,

150 ktg of proteins were separated on a 15 96 SDS gel, blotted onto a PVDF membrane, and probed with

an antibody against cytooltrorne c, as described in Materials and methods.

Control cells had an elongated shape (I‘i 1 g. 23a), and poorly bound AV (Fig. 23b). Thirty

min after incubation with 10 l&I TPP& and illumination for 2 min HV3 cells showed

extensive membrane blebbing (Fig. 23~) and increased AV staining (Fig. 23d) in the

absence of PI staining (not shown). Cellular changes were similar when cells were

incubated with 0.1 1lM mTHPC tbr 24 h and illuminated for 1 min (not shown). Figs.

23e and f show HV3 cells 30 min after incubation with 1 ;IM VU-HO and illumination

for 6 min. Cells were rounded off, membrane blebbing and AV binding were slightly

increased as compared to control cells. Damage was more pronounced with time. I322

cells 1 h after incubation with VI3-RO and irradiation were already disintegrated (Fig.

23g), bound to AV (Fig. 2311) and were positive for PI (Fig. 23i). Despite slight cross-

talk between PI and PS Uuorescence, PI staining could clearly be identified since none

of the PS localised in the nucleus.

Fig. 23, Membrane blebbiltg and AV binding of B22 and BV3 cells after incubation with pltotosensitisers

and illumination. Cells WI-C stained \vith A\‘ and PI. a. c. e, g: bright field images, b, d, f, II: AV

fluorescence, i: PI fluo~escettce. Typical pictures (750 ;un s 250 Ann) of I322 contxol cells (a, b), BV3

cells 30 min after incubation with 10 ~11~1 ‘I‘PPS.I and illimtiitation for 2 min (c, d). RV3 cells 30 tttin after

incubation with 1 !&I VB-130 and ilIumination for 6 min (e. f) and B12 cells 1 h after incubation with

VB-BO and illuntinatiott (9. 11. i) are slto~vn. Arro\vlteads indicate ntemblbratte blebbing (c) or cells

rounding off(e).

65 7. Results

66 7. Results

7.3.5. Dark toxicity and light-induced toxicity of AzE in BV3 and B22 cells

A$ is a lipophilic cationic fluorophore present in lipofbscin granules. As

determined by fluorescence microscopy after 24 h incubation time, AZF, localised in

BV3 and B22 cells in organelks resembling mitochondria and/or lysosomes (Fig. 24).

Fig. 24. Localisation of A?E fhmrescence in BV? and B22 cells. BW and 1322 cells were incubated with

10 @I AIE: for 24 h. Fluorescence n-as excited with a -150,/490 mn bandpass Clter, and detcctecl with a

5 15 mn longpass filter. a, c: autofluoresocnce of BV3 anti B22 cells, respectively. b, d: A2E fluorescence

in BV3 and 022 cells, respectively. Picture size is 220 pm s 175 pi.

Fig, 25 shows dark toxicity ofA?E in RIT3 and B22 cells. Both cell lines showed

coniparal3lc sensitivity.

Uhuninntion ofBV.3 and B21 cells slmved an additional rather than a synergistic

effect with incubation with AzE (Fig. %a). Since this is probably due to a poor

absorbance of AlE of the white light emitted by the xenon lamp, a filter for blue light

67 7. Results

was used (Fig. 2619. Also under these conditions no synergistic effect on cell survival ol

light and AZE was observed. This study \vas not followed fixther since no qpropriatc

light source was available.

1.2

1

iJ 0.8

:: Fig. 25. Dark toxicity of h,E ill BV3 and B22 cells. M 0.6 .% Cells were imubated with the indicated comem

‘E 0.4 i;?

hations for 24 11 in the dark. Cell survival was

0.2 detemined by MTT assay. BVJ cells, B22

cells. 0

0 25 50 75 100

.A,E concentration @Xl)

00 0 2 4 6 8

illumination time (niin)

I I I I 1

0 2 4 6 8 ihninatiori time (min)

68 7. Results

7.3.6. Uptake and dark toxicity of VB-BO in N4 17 cells

‘To fbrther test the hypothesis that Bcl-2 overexpressing cells show enhanced

sensitivity to lipophilic cationic PS due to higher ilLI’ Andy therefore, increased uptake of

these compo~mls, three cell lines derived from $4417 cells with different levels of Bcl-2

expression (control, Bcl- 2*2. Bcl-2*3) were used. liptake of VB-BO into control N417

cells was slightly higher than in Bcl-2 overexpressing cells, although the difference was

statistically not signiticant (Fig, 27).

250

0 control B&2*2 &l-2*3

69 7. Results

Dark toxicity of VB-BO in N417 cells was also determined. In contrast to BV3 and B22

cells, N417 cells grow in suspension. Therefore, the MTT assay had to be modilied. As

a consequence of this, lower concentrations of VB-BO and a longer incubation time

were used. Fig. 28 shows dark toxicity of VB-BO in N417 cells. In contrast to the

results with BV3 and B22 cells reported above, Bcl-2 overexpressing N417 cells were

less sensitive to VB-BO. Bcl-2*2 cells showed a higher survival compared to control

cells when incubated with 0.3 11.M VR-BO for 20 11. Light-induced toxicity of VB-BO

could not be determined, since N417 cells showed a high sensitivity to white light even

in the absence of a PS.

+ control --Cb-B&2*2 - A- &I-2"3

1 X-08 l.E-07 1 K-06 l.E-05

VU-BO concentration (M)

Fig. 28. Dark toxicity of Vl3-130 in X417 cells. Cells wxe incubated with the indicated concentrations for

20 h, and survival was determined by MTT assay. II---~, * statistically significant difference (control VS.

Bcl-2*2, a-0.05).

70 7. Results

7.4. B&2 protects isolated mitochondria but not cells j?rom ceramide-

induced effects

111 a study with isolated mitochondria, we showed that N-acetylsphingosine (C2)

and N-l?e~anoylsphingositlc (C6) ceramide and, to a much lesser extent, dihydro-

ceramidc (DHC) induce cytochrome c release. This could be prevented by addition of

hriman recombinant Rcl-2. Upon cytochrome c loss, mitochondl-ial oxygen

cotmmption, AY and Ca”. rctcntion were diminished. It was investigated whether Rcl-

2 overexpressing cells were protected against ccrarnide-induced cell death. Fig. 29

shows survival of B22 and BV3 cells incubated with increasing C2, C6 or DHC

concentrations.

0 25 50 75 100 C2 concentration (@I)

0 25 50 75 100 III-IC concentration (p\l)

0 2s 50 75 100 C6 comxntration (FM)

71 7. Results

The order of toxicity was: C6 > C2 >> DHC. Althotlgh single experiments

indicated protection by Bcl-2 of ceramide induced cell death (not sl-~own), on average no

significant differences between BV3 and B22 survival were fomld.

To determine whether ceramide would induce apoplotic ccl1 death in RV3 and

B22 cells, they were stained with AV and PI. FI,. ‘0 30 shows BV3 cells inmbated with

50 pM C2 for 30 min. Enhanced binding of .4V to cells incubated with C2 (60 % AV

positive cells) compared to control cells (26 ?,a) was found. When cells were incubated

with 100 M C6 for 2 h, they did not only show AV binding but also mcleav staining

with PI, indicating either late apoptosis or necrosis (not shown). No differences between

BV3 and B22 cells were seen.

I:ig. 30. ,k\’ binding to BV3 cells ntkr incubation with CL. 13V3 cells were incubated with 50 pM C2 for

30 min and stained with AV and PI. Fluorescence was detected by CUM. a. b: control cells. c, d: cells

incubated with C2. a, c: AV fl~m~scem~. b, d: PI fklorej~ence. Picture six is 250 pm x 250 pm.

72 7. Results

7.5. Skirt photosensitivity in PDT

7.5.1. Protective substances

The efficacy of several protective substances to reduce transmission of laser

light o-f 652 nm had been detemined. The findings were to be confirmed with an i/l

I&O model consisting of human keratinocytes, mTFIPC as the PS, and a xenon lamp

mimicking sun light. Fig . 3 1 shows the survival expressed as % CF of cells incubated

with m’IWPC and illuminated directly or tlmugh a layer of the protective substances

indicated.

I 1 100 * #

80

60

40

20

0

Protective substances against skin photosensitivity. HaCaT cells were incubated with 0.1 11~

mTI-IPCInll for 24 h and illuminated during 1 rnin (3.62 J!crn”). When indicated, 2 mg/cm of the

protective substance mxs applied on ‘Trampore tape through which the light xvas administcr.ed. Results are

presented as means -!- SE of 3 independent experiments. K siqificant differencc (substance IU.

‘I’ransporei, * significant diffcrcnce (substances as indicated). Statistical analysis was done with a one-

sided Wilcoxon-hlanu-~Vhitll~~ test. n-0.05.

73 7. Results

35 O/o Ti02, 9 ‘56 ‘IX02 --t 1 % Fez03, and light and dark cover cream protected -_

HaCaT cells significantly from m’THPC-mediated light toxicity. Addition of 1 041 FezO;

increased the protective effect of ZnO and TiOl. Dark co\er cream was more protective

than light cover cream and was the best protective substance tested.

7.52. Hyperiforce, a Hypericum extract, displays characteristics of a PS

Prompted by a case report of a woman

showing severe skin reactions upon intake of

hyperiforce and ALA, we sought to investigate a

possible synergistic effect of these two drugs,

Hyperiforce contains photosensitising cornpou~~ds

that fluoresce and accumulate in HaCaT cells mostly

in membranes and perinuclear organelles when cells

are incubated with 1 %O hyperiforce (v/v medium) for

24 h (Fig. 32).

Dark toxicity of hyperiforce was determined

by a CF assay. It exceeded 10 % for hyperiforce

concentrations above 1 ‘$60 (v/v medium) and 99 04

for concentrations above 6.25 960 (Fig. 33).

Fig. 32. Hyperifcmc fluorescence in HaCaT cells. HaCaT cells

were incubated for 24 11 with 1 960 hyperiforce. Fluorescence

was excited with a 4501400 nm bandpass filter, and detected

with a 515 nm longpass filter. a: aulotluorescencc of IIaCaT

cells. b, c: I1yperiforoc tluoKscrnce.

74 7. Results

kz 100 '3 g 8o ;r 60 2 0 2 40

s 20

0 0 0.125 0.25 0.5 0.75 1 1.25 1.875 2.5 6.25

o/o0 liyperiforce (v/v)

Fig. 33. Dark toxicity of lryperiforce. HaCaT cells were incubated with increasing concentrations of

hyperiforce fohr 24 hi and survival \vas detemined by a CF assay. Values are means from one experiment

3: SE,

Incubation of HaCaT cells with 1 mM ALA for 3 11 resulted in no dark toxicity,

and only minor light-induced toxicity was observed (Fis , 34). Incubation of the cells

with 1 960 llypcriforce for 24 11 resulted in 5 % reduction of cell survival in the dark, and

illumination time (min)

75 7. Results

upon illumination the CF was further diminished. Incubation with AL,A and hyperiforce

in the dark and LIPOII illumination resulted in a synergistic effect of these two

compounds> i. e.) CF was lower than expected from adding up toxicity of both

con~poLnl~ds alo11e.

76 8. Discussion

8.1. Mitoclzordria as a target qf m THPCphotoserlsitisation

8.1.1. Localisation of mTHPC

There is no doubt that mitochondria are sensitive targets in PDT. Lipophilic, and

especially cationic, PS accumulate at least partially in mitochondria (Woodburn et nl.,

1991). Although confocal laser scanning microscopy showed a diffllse cytoplasmic

localisation of HpD in (16 and V79 cells (Woodbum et (I/., 1991), mitochondrial

enzymes were inhibited by HpD-medi ated PI)?‘ of R3 230AC mammaty

adenocarcinoma cells in contrast to cytosolic enzymes, which remained largely

unaffected (Hilf et Al., 1984). Also the most effective PS (in terms of cell killing) in a

group of porphyrins tested were the ones localising in mitochondria (Woodburn et ul.,

1992). mTHPC is a neutral lipophilic PS that localises in membranes and organellcs

around the nucleus of MCF-7 and V-79 cells (Hornung et al., 1997; Fig. 10). By double

staining with Rh123 we could show that some of these organelles correspond to

mitochondria (Fig. 11). In human sperm cells mTHPC also accumulated in

mitochondria, that are located in their midpiece (Fig. 12). Additionally, in V~IJO mTHPC

accumulated in mitochondria as measured in liver mitochondria of mice injected with

rnTHPC 48 11 before (Fig. 9, Table 6). Not considerin g excretion of the PS, mTHPC

accmmulation was 2- to 3-fold in liver mitochondria (0.55 to 0.9 ng mTHPC/mg protein)

as compared to the concentration cspected for a ulnifbrm distribution in all tissues (0.3

rng mTHPC/kg body weight injected). In a study by Peng et 111. (l!?IS), mice were

illjected with 1 mg/kg body weight mTHPC, and concentrations of mTHPC in various

tissues were determined at different time points. Highest concentrations were found in

the urinary tract, skin, tumour (mammary carcinoma) and liver, and were about 3 and

1.2 ng/mg in liver (24 and 38 h after injection, respectively). Although more

experiments are needed to support these tIndings, these data su ggest an accumulation of

mTHPC in liver mitochondria as compared to total liver or other tissues.

77 8. Discussion

8.1.2. Yhotosemitisation of isolated mitochondria

%)amage to isolated rat liver mitochondria by deuteroporphyrin as a F’S &s

irradiation was shown by Sandberg and Romslo (1980). They found the following

sequence of reactions: uncoupling and inhibition of oxidative phosphorylation,

decreased AY, inhibitiot~ of respiration, swellin g, and disruption of mitochondria. These

findings correspond quite well with our results for PDT using mTHPC: Already at a low

m’THPC concentration mitochondrial oxygen consumption was no longer stimulated by

ADP addition (Fig. li), although at this concentration the mitochondria were still able to

build LL~ an, albeit reduced, AY’ (Fi g. 7). This is in agreement with the study of &let et

nl. (1991) who observed that AY was only slightly affected at a PDT dose that

completely abolished coupling, and concluded that uncoupling was not simply a

consequence of membrane damage. At the lowest mTHPC concentration we used,

oxygen consumption supported by pyruvate and malate was stimulated after irradiation.

also indicating uncoupling (Table 5). Similar results Jvith l~aematoporpl7yril1~pllyril~ as the 1%

later confirmed our findings (Salet et nl., 1998). Inhibition of oxidative phosphorylation

has been explained by noncompetitive inhibition of the adenine nucleotide translocator

after photofrin @-mediated PDT (Atlante tzt al., 1239). Dissipation of AY and

inhibition of I-cspiration were found when higher mTHPC concentrations were present

dunkg irradiation. Increased respiration of complex I after a mild assault using

haematoporphyrin as a PS had been previously observed (Salct and Moreno, 198 1; Salet

et nl. i 1983). Impaired Ca” homeostasis of pllotodyllamically treated mitochondria was

reported by the same group, which is in accordance bvith our study (Fig. 8). Later it was

suggested that oxidatil-e damage to mitochondria induced by PDT also includes

inactivation of the tnitochondrial permeability transition pore, possibly by the

degradation of critical histidines (,Salet ef nl. Y 1997).

78 8. Discussion

8.13. Photosensitisation of MCF-7 cells

Following the investigations on isolated mitochondria described above, we were

i&crested in the cluestion whether mitochondria are the prime cellular targets in the

photosensitisation with mTHPC. When MCF-7 cells were incubated with mTHPC and

irradiated, oxygen consumption in a pyruvate-containing medium in the absence or

presence of an uncoupler was diminished compared to control cells (Fig. 13). A

dccrcase to 69 o/o of the control values was observed under conditions that yield only 0.X

% colony formation (Table 7). However, it should be consider& that a decrease in oxy-

gen consumption, as seen with isolated mitochondria, reflects mostly oxidative damage

to the proteins of the respiratory chain, and is not the most sensitive parameter to deter-

mine impairment of mitochondria. A’+’ was also diminished in MCF-7 cells after incuba-

tion with mTHPC and irradiation (Fig. 14)? but was not quantitatively assessed. Thus,

mitochondria seem to be a target in mTHPC-tnediated PDT, but possibly not the only

target, which is expected considering the subcellular distribution of mWPC in various

organelles and membranes. Similar results were also reported for another dye, mero-

cyanine 540, that is preactivated by light before addition to the cells. During incubation

with the PS, MCF-7 cells released Rh123, with \\%icl-t they had been loaded, and ATP

levels and the activity of succinate dehydrogenase decreased (Gulliya et nL.> 1995). As

discussed above, the lipophilic mTHPC accumulates mainly in membranes, but not in

the nucleus. Thus, it is not astonishing that after mTHPC-mediated PDT of human

mycloid leukemia cells no DNA damage was detectable using a comet assay (McNair et

nl., 1997). The integrity of the plasma membrane, ho\yever, was diminished as shown

by the release of lactate dehydrogenase from normal fibroblastic and murine hepatoma

cells (Kirveliene el (zl., 1997). Damage to mitochondria can also be measured by MTT

assay, that reflects activity of mitochondrial oxidoreductases. After incubation of RI-K

and MH22 cells with 0.5 pg/ml m?‘HPC and irradiation, MTT activity decreased during

5 h after irradiation. Interestingly, the ATP level in these cells decreased only in a

medium depleted of energy sources, but not in a glucose-containirlg medium, where the

cells seemed to compensate the failure of mitochondrial ATP production by enhanced

glycolysis (Kirvelicnc cl nl., 1997). Similar results had been reported for HpD-mediated

PDT (Hilf et nl., 1986), and fLirther support the sensitivity ofmitochondria in PDT.

79 8. Discussion

8.2. O-xidatiw damage by mTHPC-mediated in vitro PDT

When isolated rat liver mitochondria were incubated with rnTHPC and

subsequently irradiated, the amount of carbonyl gro~~ps and TBARS was significantly

enhanced, dependent on the mTHPC concentration present (Figs. 4 and S). Presumably

as a consequence of these oxidative modifications, mitochondrial fimctions were

diminished as discussed above.

The use of scavengers of different oxygen radicals is one possibility to

distinguish whether a photosensitiser reacts sj~l a type I or type II mechanism (@otti>

1990). Since the addition of 1,3-dipl~enylisobe~IzofL~~al~ before irradiation reduces cell

inactivation during PDT, mTHPC is thought to act partially via type II reactions (Ma e2

rrl., 1994). We also observed a decreased formation of LOOH in the presence of DMF,

indicating type II reactions (Table 4). This is supported by the finding that LOOH

formation was increased when the incubation and irradiation took place in DzO instead

of I+O. On the other hand, BHT reduced the amount of all oxidative modifications

measured, therefore type I reactions could also play a role in r_ulTF-IPC-rllediated PDT.

However, as discussed by Girotti (1990), there are several limitations in using

scavengers of reactive oxygen species to investigate the reaction type, since many of

them lack absolute specificity. According to theoretical considerations, TBARS should

not be found as a result of type 11 reactions, but in the presence of iron decomposition of

LOOH generated may occur and lead to TBARS formation, i. e.? low amounts of LOOH

and high amounts of TBARS do not rule out the involvement of type II reactions.

StudyinS oxidative damage in MCF-7 cells, increase in CF rather thau oxidative

modifications were measured in order to determine whether a given antioxidant had an

effect (Fig. 15). Although mannitol showed the strongest ability to increase CF, the data

still suggest a mixed type I/type II mechanism. In contrast to these experiments,

mannitol had no effect on oxidative damage in isolated mitochondria that were

incubated with rnTHPC and irradiated (Table 4). The minor increase in CF (10 to 20%,)

seen here when using antioxidants could be explained by the I-inding that cells are able

to repair damage such as lipid peroxidation induced by PDT (Kirveliene et nl., 1997).

80 8. Discussion

8.3. Protection against and sensitisation to in vitro PDT hy over-

expression of Bcl-2

8.3.1. Enhanced sensitivity of’ Bcl-2 overespressing L929 cells to the lipophilic

cationic photosensitiser Victoria blue BO

The sensitivity of control (BV3) and Bcl-2 overexpressing (B22) LO29 cells to

three PS, alone or in combination with light were investigated. It is known that

overexpression of Bcl-2 or Bcl-xl, can protect cells against PDT (He et al., 1996;

Granvillc et al., 1998), whereas transfection with bcl-2 antisense oligonucleotide

increases the sensitivity of cells to PDT (Zhang cl nl., 1999). Here we show that,

depending on the PS used, 13~1-2 expression has a positive, none or even a negative

effect on survival of cells after in vitro PDT.

TPPSJ, a hydrophilic anionic PS, accunulated in organelles resembling

lysosomes (Fig, 1 SC and t), was taken up equally into both cell lines (Table 8). and

displayed equal dark toxicity to BV3 and B22 cells (Fig. 2 la). When incubation with

the PS was followed by illumination, BV3 cells had a lower surviving fraction than I322

cells (Fig. 21b). mTHPC, a lipophilic neutral PS, was localised in membranous

structures (Fig. 18b and e), and was also taken up equally into both cell lines (Table 8).

Overexpression of 13~1-2 in 1322 cells had no effect on the level of toxicity of the PS

alone or in combination with light (Fig. 20). In contrast, Bcl-2 overexpressing cells

showed an increased uptake of the lipophilic cationic PS VB-HO (Table S), that

accumulated in mitochondria (Fi g. 1 Sa and d). Accordingly, they were more sensitive to

VR-BO in the dark (Fig. 19a and b) and combined with illumination (Fig. 1%). The

enhanced sensitivity of B22 cells to VB-BO is specific, B22 cells are not generally more

sensitive than RV3 cells as shown by their equal or diminished sensitivity to mTtlPC

and TPPSJ. phototoxicity, respectively.

The antiapoptotic protein Bcl-2 inhibit-s cytochrome c release from mitochondria

(Kluck et nl., 1997), but it is not fGlly understood if and how Bcl-2 affects AY in cells

not undergoing programmed cell death, e. s., in the Bcl-2 overexpressing 1,929 cells

used in this study. Smets et al. (1994) investigated several leukemic cells with different

81 8. Discussion

sensitivity to glucocorticoid-induced apoptosis. The cells with highest resistance had the

longest cell cycle, highest content of Bcl-2 and highest ATP levels, which could reflect

increased mitochondrial activity or tighter coupling resulting in increased AY. This may

be due to the ability of Bcl-2 and related proteins to form ion channels (Schendel et ul.,

1997; Minn et nl., ‘1997; Antonsson el nl.. 1997). A study with isolated mitochondria

gave evidence that Bcl-2 regulates proton flux, since mitochondria with an elevated Bcl-

2 content were able to maintain A\f’ by enhancing proton efflux upon addition of AY-

loss-inducing stimuli, e. g., Cal+, a protonophore, or valinomycin (Shimizu et al., 1998).

Addition of human recombinant Bcl-2 to isolated rat liver mitochondria can increase

AY as measured with the probe safi-anine (unpublished observation).

PDT kills cells via necrosis or apoptosis, depending on the PS, cell type and

treatment protocol (Dcllinger, 1996; Luo and Kessel, 1997). When mitochondria arc the

subcellular target, PDT induces apoptosis (Ball et (II., 1998; Noodt et nl., 1998; Kessel

and Luo, 1999), but for lysosomal targeting contradictory results were reported (Brunk

et al., 1997; Kessel and Luo, 1998). It has been shown that various PS can elicit

different apoptotic pathways depending on their intracellular localisation (Noodt et al.,

1999). In our study, analysis of AY loss or stabilisatiorl upon PDT was technically not

possible because of interference of PS fluorescence with probes for FACS analysis.

Therefore, AV binding, a marker of apoptotic cell death, was qualitatively determined

by confocal laser scanning microscopy (Fi,. -_ (7 31). Enhanced AV binding and extensive

membrane blebbing indicate the onset of programmed cell death in both cell lines and

with all three PS used. However, lipophilic PS are likely to target the plasma membrane

resulting in blebbing independent of the mode of cell death. ‘Therefore, we additionally

investigated cytochrome c release from mitochondria (Fig, 32), another hallmark of

apoptosis known to 0cc.111' after PDT (Varnes et nl.? 1999). Cytochrome c release is

expected to happen when mitochondria are a target of the PS (Kessel and Luo, 1099), as

for VB-I30 in our study, where mitochondria are the exclusive, or for mTHPC, where

they are one of the targets. As expected, when incubated with VB-BO and illuminated,

B22 cells showed a marked cytochrome c release in contrast to BV3 cells despite the

higher Bcl-2 content ofB22 cells (Fig. 22 ). B22 and Bt73 cells were equally sensitive to

mTHPC-mediated toxicity (Fig. 30). Cvtochrome c release from 1322 cells was less i

pronounced with the PS mTHPC (Fi g. 22) than with VB-HO, although conditions for

PDT with VB-BO and mTHPC were used that induced comparable cell death in I322

82 8. Discussion

cells. XJpon illumination, reactive oxygen species are formed at the sites of mTHPC

localisation, including cell membranes. Initial apoptosis induced by PDT can be delayed

or stopped when the cell membrane is destroyed, because factors of the apoptotic

cascade are lost from the cytosol (Kessel and Luo, 109s; Kessel et nl., 1997)> and it

might be interesting to study later events in apoptosis upon mTHPC-mediated PDT.

B22 cells were more resistant than BV3 cells to TPPSd phototoxicity mediated via

lysosome destruction (Fi g. 2 I), although in B22 cells cytochrome c release was slightly

higher (Fig. 22).

In a very recent study, it was found that overexpression oEBcl-2 was paralleled

by enhanced expression of Bax in MCFlOA cells (Kim et nl., 1999). PDT with

aluminium pl~tllalocy~~t~ine selectively destroyed Bcl-2, resulting in an enhanced

apoptotic response in the cell line transfected with Bcl-2. This is supported by the

finding that also in chemotherapy-induced apoptosis Bcl-2 cleavage is part of the

apoptotic cascade (Fadecl ct crl., 1999). It cannot be excluded that Bcl-2 was destroyed

when I322 and BV3 cells were incubated with VB-HO and illuminated. However, there

are several differences in the study by Kim et nl, (1999) and ours, so that direct

comparison is difficult. Firstly, aluminium phthalocyanine is localised diffusely in the

cytoplasm (Luo and Kesscl, 1997) whereas \‘B-BO accumulates in mitochondria (Fig.

18a and d). Secondly, control and Bcl-2 overexpressing MCFlOA cells took up equal

amounts of aluminium phthalocyanine, whereas B22 cells took up more VB-BO than

BV3 cells (Table 8). The difference in PS uptake is believed to be the reason for

enhanced killing of B22 cells by incubation with VB-BO, but destruction of Bcl-2

resulting in enhanced apoptotic response may also have taken place.

The B22 cells were used as a model for cancer cells with which they share two

characteristics: they are protected against apoptosis, and they sl~~w higher AY

(Nadakavukaren et ni., ‘1985) as compared to normal cells, or WV3 cells in this study.

Lipophilic cationic drugs, e. g., VB-BO, accumulate in response to AY’ preferentially in

tumour cells and may display high toxicity despite the increased resistance of the cells

to other stress factors. It remains to be investigated whether this promising approach is

also a successful strategy for targeting and killing cancer cells i,l G\Y~.

Small cell lung cancer cells (N417) with different levels of Bcl-2 expression

were also used to further support our hypothesis. However, the Bcl-2 overexpressing

N417 cells took uy, less VB-BO (Fi,. * 27) and were, accordingly, less sensitive to this PS

83 8. Discussion

in the dark (Fig. 28). Light-induced cell killing could not be determined, since these

cells were very sensitive to light even in the absence of a PS.

8.3.2. Effects of ceramide

Many pathways are elicited by PDT that finally contribute to (apoptotic) cell

death, amongst the formation of the second messenger ceramide (Separovic ct ~1.) 1997;

Separovic et nl., 1998). 111 a study with isolated rat liver mitochondria, we observed that

ceramide induced cytochrome c release resulting in decreased mitochondrial functions?

and that these effects were inhibitable by Bcl-2. The Bcl-2 overexpressing B22 cells are

more resistant to tumour necrosis factor-a-mediated cytotoxicity than BV3 cells

(Hennet et al., 1993). Since tumour necrosis factor-a receptor stimulation induces (-le

uovo formation of ceramide, B22 cells were expected to be also protected against

ceramide-mediated cytotoxicity, as is reported in the literature for other cells

overexpressing Bcl-2 (Zhan g et al., 1997). However, although ceramide induced an

apoptotic response in BV3 and B22 cells as shown by enhanced binding of AV (Fig.

30), no significant differences in cell survival upon incubation with C2, C6 or DHC

were found (Fig. 29). This might be due to the finding that Bcl-2 not only prevents c

cytochrome c rclcase from mitochondria, but also inhibits ceramide formation

(Yoshimura et r~l., 1998). Thus, Bcl-2 might be twofold protective against tumour

necrosis factor-a but not ceramide, and, therefore, its effect on cell survival would be

more pronounced when tumour necrosis factor-a is the cytotoxic agent.

8.3.3. A$, a component of lipofuscin, as a photosensitiser

N7ith age, lipofuscin granules accumulate in retinal pigment epithelial cells of

humans (Feeney-Burns c:t al., 1980). One of the major -fluorophores present in

lipofuscin is AzE? a lipophilic cationic compound formed of two molecules of retinal

and one molecule of ethanolamine (Sakai et al,, 19%). LipofLlscin has properties of a

84 8. Discussion

PS, namely upon light absorption formation of ‘02 occurs (Gaillard et nl., 1995). AzE

coupled to LDL has been found to accumulate in lysosomes of human retinal pigment

epithelial cells and to inhibit lysosomal functions (Holz et nl., 1999). Since ,4zE is a

lipophilic cationic compound, one would expect it to be taken up by mitochondria. Our

microscopy study can neither conlkm nor discard this hypothesis. The observed

fluorescence in BV3 and B22 cells could be attributed to mitochondrial or lysosomal

structures (Fig. , 24). B22 and BV3 cells shelved no sigrkficant difference in sensitivity

towards AzE, although BV3 cells seemed to be slightly more sensitive (Figs. 25 and 26).

Only a minor light-induced effect by A2E on cells \vas observed (Fig. 26). It was

impossible to illuminate the cells for longer periods of time, since light alone in the

absence of a PS already reduced cell survival under these conditions. The emission

spectrum of the xenon lamp (400 to 780 IIKI) does not we1 1 correspond to the absorbance

spectrnm of A$ (absorption maxima at 336 and 436 nm). However, with an appropriate

light source AzE-mediated phototoxicity is very likely to occur, since irradiation with

blue light of lipo&scin-loaded cells has been shown to decrease cell viability and

lysosomal stability (Wihlmark et nl., 1997).

8.4. Role qf Ca” and NO in PDT

When the plasma membrane is a target of PDT, as in 17l’T’T-fPC-mediated PDT.

lipids and proteins will be oxidatively damaged. As a result, the membrane becomes

permeable for ions, e. g. Ca”. When mouse tnyeloma cells were photosensitised with

zinc phthalocyanine, plasma membrane depolarisation was observed (Specht and

Rodgers, 199 I ). This depolarisation depended on the cxtracellular concentration of Na+,

was diminished in the presence of a Na-*- channel blocker, and was accompanied by an

increase in cytosolic Cal” concentration. After aluminium phthalocyanine-mediated

PDT, Chinese hamster ovary cells transiently had increased cytosolic Ca2’

concentrations (Penning et &., 1992). This response was only eliminated in the presence

85 8. Discussion

of 1 mM EGTA in the medium, but not by 20 PM verapamil. Interestingly, chelating

intracellular Ca” enhanced cell killing, indicating a role of Ca2’ in cell rescue after

PDT. Similar results were found in human skin fibroblasts (Hubmer et al., .I 996).

Increase of cytosolic Ca” in human cerebral glioma cells incubated with HpD and

irradiated was due to influx of extracellular (‘a”‘. and was inhibited by the Cal channel

blocker diltiazem (Joshi et nl., 1994). When incubation wit-b the PS is long enough so

that the compound localises in intracellular organclles, cndoplasmic uptake of (:a”-‘- is

itlhibited upon irradiation which could also contribute to elevated cytoplasmic Ca”’

levels (Dellinger et nl., 1904). Influx of Ca". does not reflect a general membrane

damage upon PDT, and although it is not per* se a cause of ccl1 death (Gederaas ct al.,

1996), it can be paralleled by DNA fragmentation (Tajiri ef nl., 1998). Also in our

study, incLibation of MCF-7 cells with mTHPC and irradiation led to an increase in

intracellular (~a”-+ levels (Fig. 16).

Since the constitutive isofonnes of NOS are Cal”-dependend, we expected an

increase in NO formation following PDT. Tnterestingly. two phases o F increased nitrite

and nitrate formation were f&md (Fig. 17). This could reflect two phases of intracellular

Ca”’ accumulation, as one study reports transient elevated CL?‘.’ levels already 5 min

after- irradiation (Pemlin g et al., 1992), but another only after 1 to 2 h (T%jiri ct nl.,

1998). Alternatively, inducible NOS could be expressed. In human epidcrmoid

carcinoma cells photosensitised with a silicon phtl~alocyanine, increased expression of

constitutive NOS was found as early as 15 s after PDT (Gupta et nl., 1998). Tt remains

to be addressed - by modulation of extracellular Cal’ concentration and Western blot

analysis - which are the exact mechanisms of for the biphasic nitrite and nitrate

formation observed in mTHPC-mediated irz I&TI PDT of MU?-7 cells.

86 8. Discussion

8.5. Skin pIzotosensitivity

8.5.1. Protection against skin photosensitivity

One of the major side effects of PDT is prolonged cutaneous sensitivity. For

example, when photosensitised with photofrin, patients show skin reactions ~rpotl light

exposure up to 13 weeks after injection of the PS (Wagnieres et al., 1998). One of the

goals in the development of new PS is to reduce skin sensitivity. Skin reactions in

patients injected with the second-generation PS m’I’HPC were most pronounced 1 week

after administration, and lasted only up to 6 weeks (Wagnieres et nl., 1998). In general,

prevention of skin damage may take place at several steps in the process of

photodynamic action: i) prevention of PS accumulation in the skin, ii) prevention of

excitation of the PS by light, and iii) prevention of oxidative damage of cellular

coinponcnts.

Before our study. patients at the Iiniversity Hospital were advised to avoid

strong sunlight and to protect themelves, e. g., by using sunscreens. S~mscreens,

however, are meant to absorb light in the UV range (290 to 380 mm), but PS strongly

absorb light in the visible (380 to 780 nm) and near infrared (780 nm to 2.5 pm) range

of the spectrum. III our in ~~z’tr*o study, sunscreens showed no protection of HaCaT cells

that were incubated with mTHPC and illuminated (Fi g. 31) and only minor effects in

the following i71 viva study (by V. A. Schwarz) compared to creams containing

pigments sucl~ as TiO2 and FclO3. While the puotectivc creams tested are meant to be

applied on face and hands, special fabric materials might be used to protect other parts

of the body from visible light (Menter et crl., 1998). Many irl 11itm and irl I&O studies

have investigated the protective effect of antioxidants, c?. g., p-carotene (Moshell and

Bjotmon, 1977), N-acetylcysteine (Baas et al., 1994; Baas et al., 1995; Yusof et nl.,

1 WC)), 1,3-dipl~e~~ylisobe~~~of~~~~~~~ (McLear and Hayden, 1989), ascorbic acid (Bofh et

al., 1996), and melatonin (Zang ct ill., 1998), in order to protect patients suffering from

porphyria or undcrgoirlg PDT.

87 8. Discussion

8.52. Synergistic effects of two photosensitisers

Wypericum extracts for treatment of depression contain the photosensitising

compound hypericin and are known to induce skin photosensitivity (Golsch et nl., 1997;

Rernd el nl., 1999). Hypericin itself is used as a PS (Koren et nl., 1996), and has been

shown to induce apoptosis (Assefa et nl.. 1999). Incubation with hypericin and

irradiation of HeLa cells caused cytochrome c release and activation of caspase-3

(Vantieghem ct nl., 1998). Hypericin has been reported to localisc in the nucleus of

T47D mammary tumour cells (Miskovsky et al., 1995). In contrast, we observed

fluorescence in organelles around the ~LIC~CLIS and in membranes of HaCaT cells

incubated with hyperiforce, but never in the nucleus (Fi g. 32). More consistent with our

observations, hypericin-mediated PDT decreased cellular ATP levels and respiration in

EMT6 mouse mammary carcinoma cells, indicatin g that mitochondria were a major

subcellular target (.Iohnson et ill,, 1998).

Our aim was to study a possible synergistic effect of ALA and hyperiforce, and

to co~~firtn an observation in a patient with severe skin reactions, who had received both

drugs (in collaboration with D. Ladner). When HaCaT cells were incubated with

hyperiforce and ALA, the two PS acted in fact synergistically, i. e., the toxicity in the

dark and upon light exposure was more pronounced than expected for additive effects

(Fig. 34). In the literature, synergistic as well as antagonistic effects for the combination

of treatments with hypericin have been described. Hypericin significantly enhanced

radiosensitivity of glioblastoma cell lines (Zhang et al., 1996), but caused stimulated

growth of Staphylococcus aurcus when combined with photofrin II@ or mTHPC (Kubin

et nl., 1999).

We conclude from our data that until PS with improved properties are available,

protection against light with cover creams is a usef~tl tool for avoiding skin reactions

such as oedema and erythema in patients. Furthermore, possible photosensitising

propertics of other drugs, especially natural compounds suc~b as hypericum extracts,

should not bc underestimated.

88 8. Discussion

Selective destruction of tumour cells is the aim of anti-cancer therapies. 1.n PDT

this is accomplished by enhanced accumulation of a PS in the target tissue followed by

light activation. A variety of compounds is being tested in vitro and in viva as possible

new PS with improved properties. Amongst many parameters, the intracellular

localisation of a PS influences its capacity to kill a cell. Special attention has been paid

to mitochondria as a subcellular target, not only because they arc mainly responsible fol

energy supply in most cell types but also because their major role in apoptotic cell death

has recently become evident.

In the first part of this thesis, destruction of isolated rnitochondria or

mitochondria in MCF-7 cells by mTHPC-mediated PDT was investigated. Oxygen

consumption and AY’ of mitochondria and cells were diminished upon incubation with

m’l’HPC and irradiation. However, it cannot be excluded that damage to other organelles

contrihLttes to cell death induced by PDT with mTHPC, since this PS is not only

localised in mitochondria.

In the second part of this thesis, it was investigated whether the relatively higher

AY ol‘ carcinoma cells could be used for targetin g these cells with cationic PS. As a

model system, cells overexpressing the anti-apoptotic protein Bcl-2, that displayed

higher AY than control cells, were used. Interestingly, overexpression of Bcl-2 had a

positive, none or even a negative effect on cell survival after it1 vifm-PDT with three

different PS.

In sunmary, PDT is able to induce cell death eye11 in cells which are protected

against apoptosis or display resistance to other therapies, provided that an appropriate

PS and corresponding protocol are used.

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103 10. Acknowledgements

This work was financially supported by Leica and Avina Foundation (Switzerland).

I am very gratefLtl to:

Prof. Dr. K. H. Winterhalter and Prof. Dr. C. Richter for giving me the possibility to

ped‘orm this thesis at the Institute of Biochemistry, Swiss Federal lnstitutc of

Technology, Ziirich, and making SLY that it was coming to a good end.

PD Dr. H. Walt and Prof. Dr. II. Hailer for providin g me with a well equipped

laboratory at the Research Division of Gynaecology, Department of OB/GYN,

University Hospital Ziirich, and the possibility to follow also my own ideas.

My Parents and ‘Trudi and And& T.,ink for their continuous support.

Pedram Ghafourifar, Rosanna Cattaneo, Karin Reinhart and Martin Pruschy for helpf~rl

discussions, advices and cheering me up; Felix Margadant and Daniel Lang for

computer support; Sonia Rocha, Ursula Schuchter, B-i gi tte Jerltsch and Wolfgang

Blodig for technical help; Viola Schwarz, Christa Stahel, Nicole Filser, Gloria

Perewusnyk, Andri: Fcdier, Gisele Hiipfl, Daniela Ladner, and many others who

collaborated with me and helped me in various ways to finish this thesis.

105 11. Publications

11. PUBLTCATI'ONS

Klein, S. D,, Schweizer, M., and Richter, C. (1996) Inhibition of the pyridine

nucleotide-linked mitochondrial Ca”‘- release by 4-hydroxynonenal: the role of

thiolate-disulfide conversion. Redo.x Keporr 2, 3 53-3 58.

Klein, S. D., Walt, I-l., and Richter, C. (1997) Photosensitization of isolated rat liver

mitochondria by tetra(ru-hydroxyphenyl)chlorin. A-lr*ch. Biocl~ern. Biopl~ys. 348,

313-3’19.

Klein, S. D., Walt, H., Schwarz, V. A., and Richter, C. (1998) Mitochondria as a target

in the photosensitisation of MCF-7 cells by tetra(nl-hydroxyphenyl)chlorin

(mTHPC). Proc. 1YL4 congrms, Nantes, France.

Ghafoourifar, P., Klein, S. D., Schucht, O., Schenk, U., ~Pruschy, M., Rocha, S., and

Richter, C. (1999) Ceramide induces cytochrome c release from isolated

mitochondria. Importance of mitochondrial redox state. J. Kid. Chem. 274,

6080-6084.

Gbafourifar, I’., Schenk, II., Klein, S. D., and Richter, C, (1999) Mitochondrial nitric-

oxide synthasc stitnulatiotl canses cytocluome c release from isolated

mitocltondria. Evidence for intramitochondrial peroxynitrite formation. J, Bid.

Chem. 274, 31185-31188.

Klein, S. D., Walt, H., Rocha, S., Ghafourifar, P., Pruschy, M., Winterhalter, K. H., and

Richter, C. (2000) Enhanced sensitivity of Bcl-2 overexpressing IL929 cells to

the Iipophilic cationic photosensitiser victoria blue HO. submitted.

Schwarz, V. A,, Klein, S. D., Knochenmuss, R., Mornung, R., \;C’yss, P., Hailer, U., and

Walt, H. (2000) Skin protection for photosensitized patients. submitted.

Ladner, D. P., Klein, S. D., Steiner, R. A., and Walt, H. (2000) Synergistic toxicity of&

aminolevnlinic acid and Hypericum extract. submitted.

107 12. Congresses

12. CoNGKEssEs

Meeting of the Swiss Societies for Experimental Biology (USGER), Geneva,

Switzerland, 1997. Poster presentation: Klein, S. D., Richter, C., I-Taller, l_J., and

Walt, H. Subcellular localisation of the photosensitiser mTHPC in MCF-7 cells

and 11 nm an sp ems,

First World Congress of Photomedicine in Gynecology, Zurich, Switzerland, 1998, Oral

presentation: Klein, S. D. Photosensitization of isolated rat liver mitochondria by

tetra(rll-hydroxyphenyl)cl~lorin.

D-HIOT, Symposium of ETH Ziirich, Davos, Switzerland, 1998. Poster presentation:

Klein, S. D., Ghafourifar, P., and Richter, C. Ceramide induces cytochrome c

relcasc from isolated mitochondria.

7[” Biennial Congress of the International Photodynamic Association, Nantes, France,

1998. Poster and oral presentation, award for scientific communication: Klein, S.

D., Walt, H., Schwarz, V.A., and Richter, C. Mitochondria as a target in the

photosensitisation of MCF-7 cells by tetra(rtr-hydroxyphenyl)chlorin (mTHPC).

st” Congress of the European Society for .Photobiology, Granada, Spain, 1999. Poster

presentations: Klein, S. D., Ghafourifar, P., Walt, H., and Richter, C. Nitric

oxide formation and increase in intracellular calcium levels followitlg i/l vitro

PDT, and Schwarz, V. A., Hornung, R., Walt, T-T., Klein, S. L>., Haller, U., and

Wyss, I’. Skin protection for photosensitized patients.

109 13. Curriculum vi lae

13. CURRICULUMVITAE

Sabine Doris Klein

born I 311’ August 1972 in Z,iirich, Switzerland

citizen of Tllnau-Effretikon, ZH

1979 - 1985 Prinmy school, Effretikon, ZH

19s5 - 1991 Gymnasium, ~Kantonsschule Rychenbel,, ‘0 Winterthur, ZH, Matura

type B

1991 - 1996 Diploma in biochemistry, Swiss Federal Tnstitute of Technology

(ETH)? Ziirich. Diploma thesis at the Institute of Biochemistry,

Swiss Federal Institute of Technology, Ziirich, on “Modulation by

4-hydroxynonenal and ebsclcn of the specific mitochondrial Ca’.‘.

release” under the supervision of Prof. Dr. C. Richter.

I996 - 2000 PhD of biochemistry, Research Division of Gynaecology,

University Hospital, and Institute of Biochemistry, Swiss Federal

Institute of Technology, Ziirich under the supervision of PD Dr. H.

Walt, Prof. Dr. C. Richter and Prof. Dr. I<. H. Winterhalter

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