www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 322 (2004) 452–457

BBRC

Photosensitization with protoporphyrin IX inhibits attachmentof cancer cells to a substratum

A.B. Uzdenskya,b,*, A. Juzenienea, E. Kolpakovaa,G.-O. Hjortlanda, P. Juzenasa, J. Moana,c

a Institute for Cancer Research, 0310 Montebello, Oslo, Norwayb Rostov State University, 344090 Rostov-on-Don, Russia

c Institute of Physics, University of Oslo, 0316 Blindern, Oslo, Norway

Received 16 July 2004

Available online 10 August 2004

Abstract

Effects of photodynamic therapy (PDT) on adhesion of human adenocarcinoma cells of the line WiDr to a plastic substratum

were investigated. Protoporphyrin IX induced by 5-aminolevulinic acid (ALA) was used as a photosensitizer. Light exposure inhib-

ited attachment of suspended cells to a substratum. The adhesion was most strongly pronounced for light exposures around

200 mJ/cm2 causing cell death. However, sub-lethal exposures (42 mJ/cm2, 97% survival) inhibited cell adhesion as well. Sub-lethal

ALA-PDT increased the intracellular space in dense colonies of WiDr cells. This was attributed to formation of lamellipodia

between the cells and to increased numbers of focal contacts containing aVb3 integrin in some of the cells. The E-cadherin distri-

bution was not changed by the treatment. Complex processes, including changes in cellular shape and reorganization of the cyto-

skeleton, are suggested to participate in the observed ALA-PDT effect on the cell adhesion.

� 2004 Elsevier Inc. All rights reserved.

Keywords: 5-Aminolevulinic acid; Cell adhesion; Photodynamic therapy; Integrin; Tumour metastasis

Photodynamic therapy (PDT) is based on destruction

of malignant cells and tissues by cytotoxic reactive oxy-

gen species produced during light exposure of cells andtissues in the presence of a photosensitizer [1,2]. Among

other effects PDT damages cell membranes [3] and im-

pairs cell adhesiveness [4–9].

Adhesion is a primordial property of cells, basic for

tissue integrity and centrally involved in formation of

tumour metastasis [10,11]. The remarkable advantage

of PDT as an anti-cancer modality is its capability of

reducing tumour metastases [12,13]. This reductionmay be associated with photoimpairment of the adhe-

siveness of cancer cells [8,9]. Cell adhesion to an extra-

cellular matrix or artificial substrata is mediated by

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.07.132

* Corresponding author. Fax: +47 22 93 42 70.

E-mail addresses: uzd@krinc.ru, auzd@yandex.ru (A.B.Uzdensky).

integrins [14–16]. Integrin receptors initiate numerous

signal transduction pathways that control cell shape,

motility, proliferation, and death [16–18]. Cadherinsare responsible for cell–cell adhesion, and play an

important role in maintaining intercellular connections

and tissue architecture [14,17,19]. E-cadherin has been

shown to suppress tumour cell invasiveness and metas-

tasis [14].

5-Aminolevulinic acid (ALA), a biochemical precur-

sor of the endogenous photosensitizer protoporphyrin

IX (PpIX), is being studied for cancer treatment [20].After exogenous administration of ALA, protoporphy-

rin IX is formed and preferentially accumulates in rap-

idly proliferating cancer cells, thus providing selective

destruction of tumours exposed to light. The mecha-

nisms of ALA-PDT at the cellular and tissue levels have

been thoroughly investigated in the past decade [21–23].

A.B. Uzdensky et al. / Biochemical and Biophysical Research Communications 322 (2004) 452–457 453

However, the effect of ALA-PDT on the adhesive prop-

erties of cultured cells has not been studied in detail so

far. Recently, we investigated the effect of sub-lethal

PDT on the enzymatic detachment of cells from a sub-

stratum [24]. In the present work we have addressed

the effect of ALA-PDT on the attachment of cancer cellsto a plastic substratum, on cell morphology after attach-

ment, and on expression of integrin aVb3 and E-cad-

herin in these cells.

Fig. 1. Attachment of WiDr cells to a substratum after 2 h preincu-

bation with or without 1 mM ALA in the darkness.

Materials and methods

5-Aminolevulinic acid (ALA), RPMI-1640 medium, penicillin/

streptomycin solution, LL-glutamine, trypsin–EDTA, thiazolyl blue

tetrazolium bromide (MTT), isopropanol, phosphate-buffered saline

(PBS), and other chemicals were obtained from Sigma–Aldrich Nor-

way AS (Oslo, Norway). Foetal calf serum (FCS) was obtained from

Gibco-BRL, Life Technologies, Roskilde, Denmark. 30 mM of stock

solution of ALA in RPMI-1640 medium was prepared ex tempera

before every experiment and diluted in this medium to a final con-

centration of 1 mM. Mouse anti-human integrin aVb3 (MAB1976Z,

clone LM609) was obtained from Chemicon International (Temecula,

CA). Mouse anti-E-cadherin was obtained from Transduction Labs

(Lexington, KY). As a secondary antibody we used Cy3-conjugated

goat anti-mouse IgG obtained from Jackson Immunoresearch Labo-

ratories (West Grove, PA). Antibodies were dissolved ex tempera in

TBS (20 mM phosphate-buffered saline containing 5% (w/v) milk

powder and 0.2% (v/v) Triton X-100).

The human WiDr cell line is derived from a primary adenocarci-

noma of the rectosigmoid colon [25]. These cells were maintained in

exponential growth in RPMI 1640 medium with 10% FCS, 100 U/ml

penicillin, 100 lg/ml streptomycin, and 2 mM LL-glutamine. The cells

were incubated in cell culture flasks (Nunc A/S, Roskilde, Denmark) or

plastic 12-well dishes (Costar, Corning, Corning, NY) at 37 �C in a

humidified 5% CO2 atmosphere and subcultured twice a week using

0.01% trypsin in 0.02% EDTA.

The survival of the cells was measured using the colorimetric MTT

assay [26]. The cells were seeded in 75 cm2 cell culture flasks and grown

for three days. Then they were incubated with 1 mM ALA for 2 h,

washed with PBS, and trypsinized. Approximately 105 cells in fresh

medium were seeded into wells and exposed to light. At 24 h after

PDT, MTT (0.25 mg/ml) was added to each well and the samples were

incubated at 37 �C for 4 h. After washing and dissolving of formazan

by isopropanol (200 ll/well), 25 ll samples were transferred into a 96-

well microplate for reading of the optical density by a Multiskan MS-

352 plate reader (Labsystems, Helsinki, Finland).

The ability of the treated cells to attach to a plastic substratum (a

well bottom) was assayed by two methods. The cells were incubated for

2 h in the dark at 37 �C in a 75 cm2 Falcon flask with 1 mM ALA.

Then they were brought into suspension by the trypsin treatment. This

suspension was distributed into four 25 cm2 flasks, which were irra-

diated for 0, 0.3, 1 or 5 min (fluences 0, 14, 42 or 210 mJ/cm2,

respectively). (1) Immediately after irradiation 2 ml aliquots of the cell

suspension from each flask were added into new flask containing 5 ml

RPMI 1640 medium. This was done in triplicate for each experimental

point. Twenty-four hours after light exposure the number of attached

cells in a given area of the bottom of the flask was determined using a

microscope. (2) Immediately after irradiation triplicates of 0.5 ml

aliquots of the cell suspension from each flask were added to incuba-

tion wells containing 2 ml RPMI 1640. At 0, 1, 3 or 5 h after light

exposure 1 ml aliquot of the cell suspension was removed and the

number of non-attached, floating cells was counted by means of a

Glasstic Slide 10 with grids (Hycor Biomedical, Garden Grove, CA).

The rate of cell attachment was estimated by determining the number

of non-attached cells suspended in the medium compared to that in

control samples.

A bank of fluorescent tubes (Model 3026, Applied Photophysics,

London, UK) with an irradiance of 0.7 mW/cm2 was used for photo-

irradiation. The emission of this lamp was mainly in the wavelength

region 370–450 nm, with a peak at 405 nm.

For visualization of integrin aVb3 or E-cadherin, control and

photosensitized (1-min irradiation after 2-h incubation with 1 mM

ALA) cells grown on glass coverslips were fixed for 20 min in 3%

paraformaldehyde at 20 �C. Then they were washed three times in PBS

and labelled for 20 min with the primary antibodies dissolved in TBS,

washed twice with PBS, and then labelled for 20 min with secondary

goat anti-mouse antibodies. After staining, the coverslips were

mounted in Mowiol. Fluorescence microscopy was performed using a

Zeiss Axioplan microscope (Karl Zeiss, FRG) equipped with an oil

immersion objective 63·, an HBO/100 W mercury lamp, an excitation

filter 450–490 nm, a dichroic beam splitter FT 510, and a long pass

emission filter >630 nm. Fluorescence images were taken by a CCD-

camera (Princeton Instruments, Princeton, USA). A Leica TCSNT

(Wetzlar, Germany) microscope was used for confocal microscopy.

Images were taken with an objective 100· and captured with a reso-

lution of 1024 · 1024 pixels.

Standard statistical methods, based on Student�s criterion and the

non-parametric sign criterion, were used. Results are expressed as

means ± SEM.

Results

Suspended WiDr cells gradually sedimented on the

well bottom and attached to the plastic substratum. In

the darkness 50% of the cells were attached after

approximately 50 min. The number of attached cells

was close to maximal after 3–4 h incubation and was

constant during the following 20 h (Fig. 1). Preincuba-tion of the cells with 1 mM ALA for 2 h in the darkness

(cells were then rinsed) did not influence the cell attach-

ment (Fig. 1).

A survival curve of WiDr cells after ALA-PDT

(1 mM ALA, 2 h incubation) is presented in Fig. 2.

Short light exposures (up to 1 min, 42 mJ/cm2) were

Fig. 2. Phototoxicity of WiDr cells after preincubation with 1 mM

ALA for 2 h. The cells were exposed to light in a suspension. Data

represent three independent experiments with three parallels in each

group.

Fig. 3. Effect of different light exposure times on the attachment of

WiDr cells preincubaed with 1 mM ALA for 2 h. Data represent mean

ratios of the number of non-attached cells with the number of initial

cells in the suspension. The dynamics are averaged from 13

experiments.

454 A.B. Uzdensky et al. / Biochemical and Biophysical Research Communications 322 (2004) 452–457

practically non-phototoxic (LD10 = 50 mJ/cm2),

whereas longer exposures induced cell death (Fig. 2).

A non-phototoxic 1 min exposure (survival 97%, Fig.

2) delayed the cell attachment slightly. In the PDT

group 38% of the cells were attached compared to 57%

in the control at 1 h after light exposure (Fig. 3). Mod-

erate phototoxic light exposures (3–5 min, 126–210 mJ/

cm2) diminished the cell attachment considerably, nota-

Table 1

Number of experiments on ALA-PDT (1 mM, 2 h incubation) influence the

Time after PDT (h) Light exposure

1 min

+ 0 �1 10 2 1

5 5 5 3

‘‘+,’’ weaker attachment; ‘‘0,’’ no difference; ‘‘�,‘‘stronger attachment; and ‘

bly during the early part of the attachment period (Fig.

3). The dynamics of cell attachment were averaged from

13 independent experiments that possibly differed

slightly with respect to experimental conditions. This

might be a reason for rather large error bars in the graph

(Fig. 3). However, in spite of the variations among theexperiments, the differences between the control and

the experimental samples were significant in almost all

of the experiments. Use of the non-parametrical statisti-

cal sign criterion (Table 1) showed that the non-photo-

toxic light exposure (1 min) significantly reduced the

cell attachment at 1 h incubation.

After attachment, WiDr cells proliferated and formed

tightly packed colonies with a little space between cells(Figs. 4A, B, and E; 5A, B, and E). The integrin aVb3was diffusely distributed in these cells. Its immunofluo-

rescence was strongest in the intercellular contact regions

inside the colonies. Fluorescing points presumably indi-

cate focal contacts (Fig. 4A). ALA-PDT of the attached

cells decreased the density of cell packing (Figs. 4C, D,

and F; 5D and F) indicating an inhibitory effect on

cell–cell interactions. The cells formed lamellipodia di-rected not only to the surrounding medium, but also to

the intercellular space inside the colonies (Figs. 4D and

5D). In the photosensitized cells integrin aVb3 was redis-tributed from the intercellular regions and formed

numerous focal contacts throughout the cells. Focal con-

tacts were often observed at the leading edges of the

lamellipodia (Fig. 4C). The E-cadherin fluorescence in

WiDr cells was presented by small granules concentratedin intercellular contacts and at the outer colony border

(Figs. 5A and E). ALA-PDT did not change the distribu-

tion of E-cadherin significantly (Figs. 5C and F), except

in the border between the cells (Fig. 5F) due to the in-

creased intracellular space.

Discussion

After being seeded in an unstirred flask, the cells sedi-

ment on the bottom and attach to it. Our data demon-

strate that ALA-PDT slows down the cell attachment

process. Not only toxic, but also non-toxic light expo-

sures interfered with the cell adhesion process. Thus,

the adhesive properties are impaired even in surviving

cells. The delay of the attachment may be associated with

attachment of WiDr cells to the substratum

5 min

p + 0 � p

<0.05 9 2 1 <0.05

>0.05 11 1 0 <0.01

‘p,’’ statistical significance.

Fig. 4. Integrin aVb3 distribution in WiDr cells (A,C,E,F). In control cells integrin aVb3 localizes mainly in the peripheral regions of intercellular

contacts (A,E). After PDT (1 mMALA, 2 h incubation, 1-min irradiation) it redistributes throughout the cells (C,F). ALA-PDT apparently increases

the space between the cells in the colonies (C,F), which is filled with lamellipodia (D). (A,C) Conventional fluorescence microscopy; (B,D) phase

contrast images on the same cell colonies; and (E,F) confocal images. Bars on (A–D) 20 lm, on (E,F) 30 lm.

Fig. 5. E-cadherin distribution in WiDr cells (A,C,E,F). In control cells E-cadherin localizes mainly in the regions of intercellular contacts (A,E).

After PDT it does not significantly redistribute (B,E), but ALA-PDT apparently increases intercellular spaces in the colonies (F). (A,C) Conventional

fluorescence microscopy; (B,D) phase contrast images of the same cell colonies; and (E,F) confocal images. Scale bars for (A–D) 20 lm, for (E,F)

30 lm.

A.B. Uzdensky et al. / Biochemical and Biophysical Research Communications 322 (2004) 452–457 455

PDT-induced perturbations in the cell adhesion machin-

ery, including integrins, proteins linking integrins to the

actin cytoskeleton, and the intracellular signalling system

[6,7]. In colonies of the attached WiDr cells, we observed

morphological changes, including formation of lamelli-

podia in the intercellular space, and redistribution of inte-

grin aVb3, indicating creation of focal adhesions. PDT ofcells with hematoporphyrin derivative also decreased cell

attachment to a substratum and to a monolayer of the

same [4] or endothelial cells [5,8]. Photosensitization with

benzoporphyrin derivative-monoacid ring (BPD-MA)

inhibited cell attachment to components of the extracel-

lular matrix as fibronectin, vitronectin, and collagen [6].

It also influenced the cell shape, but, unlike our observa-

tion, inhibited adhesion-induced phosphorylation of

focal adhesion kinase [6] and caused loss of b1 integrin-containing focal adhesion plaques [7]. However, it should

be noted that the light exposures used in these works [6,7]

killed most of the cells.

Cytoplasmic structures, like mitochondria, in which

PpIX is produced from ALA [20], are not directly linkedto the adhesion machinery associated with the plasma

membrane. It is surprising that PDT targeting of mito-

chondria and other perinuclear structures by ALA-pro-

duced PpIX in the present work and in our previous

paper [24], as well as MitoTracker Red [24], or BPD-

MA [6,7] influenced the remote adhesion processes at

the cell surface where cell adhesion molecules such as

456 A.B. Uzdensky et al. / Biochemical and Biophysical Research Communications 322 (2004) 452–457

integrins and cadherins are placed [14]. One can suggest

that either a number of complex processes, including ener-

gy-dependent changes in the cell shape, reorganization of

the cytoskeleton, and signal transduction, participate in

PDT-induced impairment of the cell adhesion, or a cer-

tain fraction of the ALA-induced PpIX has been relocal-ized to the plasmamembrane at the time of light exposure.

Being receptors of the extracellular matrix, integrins

are responsible for the cell-substratum adhesion. Their

intracellular domain is linked to the actin cytoskeleton

and signalling molecules, such as focal adhesion kinase,

Src kinase, etc. [15,18,27,28]. ALA-PDT-induced

changes in the cell adhesion might be caused either by

a direct PDT effect on integrins or by a PDT-inducedmodulation of integrin expression and inside-out signal-

ling that regulates integrin-ligand affinity and clustering

necessary for strong adhesion [28,29]. Margaron et al.

[6] showed that even strong photosensitization of cancer

cells with BPD-MA (10% survival) did not affect the

expression level of diverse integrins with a2, a4, a5, aV,b1, b2, and b3 subunits. Therefore, the PDT effect on cell

adhesion may be associated with regulation of the affin-ity and/or of the clustering of existing integrin molecules

rather than with their degradation or inhibition of their

expression. We observed redistribution of integrin aVb3and formation of focal adhesions. Simultaneously lamel-

lipodia formation, indicating weakening of intercellular

interactions in the colonies, was observed. However, the

distribution of E-cadherins (responsible for intercellular

contacts [14]) was not changed. Therefore, E-cadherinswere probably not involved in the changes in cell mor-

phology and adhesion.

The aVb3 integrin is a major integrin in focal contacts

[15,28,30]. It binds specifically to vitronectin, a serum

protein that triggers cell attachment [31]. This high-af-

finity integrin plays an important role for formation of

new adhesion points and promotion of lamellipodia,

for cell proliferation, survival, migration, invasion, andfor formation of tumour metastases [30,32]. One can

suggest that a residual amount of a photosensitizer is re-

tained in the plasma membrane during influx of PpIX

after extracellular application or efflux in the case of

ALA-PDT [24] may be sufficient for inducing lipid per-

oxidation [3] and protein cross-linking in the plasma

membrane [33]. This may prevent conformational tran-

sition of integrins to their active state [29] and hindertheir clustering necessary for adhesion [15,29]. ALA-

produced PpIX might also sensitize inside-out signalling

proteins such as Rac, Rho, protein kinase C, etc., that

influence integrin binding to the substratum ligands

and control formation of lamellipodia, stress fibres,

and general cellular response, respectively [15,27,32,34].

A decrease in the adhesiveness of cancer cells after

PDT may reduce the metastatic potential of survivingtumour cells [8,9]. PDT-induced inhibition of tumour

metastasis has been reported in a number of papers

[12,13,35,36]. This may be a unique advantage of PDT

over other cancer treatment modalities, and may be re-

lated to PDT-induced inhibition of both cell attachment

to extracellular matrix or other cells and to changed

detachment of adherent cells [24].

Acknowledgments

The present work was supported by the Research

Foundation of the Norwegian Radium Hospital and

by the Norwegian Research Council.

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