Phorochemisrrj and Photobiology Vol. 46. No. 5 . pp. 651-656, 1987 Printed in Great Britiiin. All rights reserved

0031-8655187 $03.00+0.00 Copyright Q I087 Pcrgamon Journals Ltd

PHTHALOCYANINE PHOTOSENSITIZATION OF MAMMALIAN CELLS: BIOCHEMICAL AND

ULTRASTRUCTURAL EFFECTS

E. BEN-HuR'", M. GREEN'. A. PRAGER', R . KOL' and I. ROSENTHAL? 'Nuclear Research Center-Negev, P.O. Box 9001, Beer-Sheva 84190, Israel, 'A.R.O. , The Volcani

Center, P.O. Box 6 , Bet-Dagan, Israel

(Received 11 June 1987; accepted 17 June 1987)

Abstract-The incorporation of thymidine, uridine and leucine into DNA, RNA and proteins, respect- ively, was measured in logphase Chinese hamster cells photosensitized by chloroaluminum phthalo- cyanine tetrasulfonate (AIPCS). Post-treatment synthesis of all macromolecules was inhibited. The inhibition became progressively more pronounced with time, reaching a maximum at ca. 3 h after treatment. The differences between relative sensitivity of protein, RNA and DNA syntheses to AlPCS photosensitization, were not statistically significant. Some of the observed inhibition was due to a reduced uptake of the labeled precursors from the growth medium. Energy metabolism, as reflected by glucose oxidation, was sensitive to AIPCS plus light. Inhibition of glucose oxidation was evident immediately after treatment, and became more pronounced with time. Following a sublethal light fluence, maximum inhibition was observed at 3 h and there was a gradual recovery at later times. Inhibition of glucose oxidation was about two fold higher in plateau-phase compared to log-phase cells. The former were also twice as sensitive with respect to cell killing. These results suggest that inhibition of glucose oxidation induced by mitochondria1 damage as seen in human lymphocytes, may be a primary cause for AIPCS-photosensitized cell killing.

INTRODUCTION

Some phthalocyanines (PC)t are efficient photosen- sitizers for mammalian cells in vitro (Ben-Hur and Rosenthal, 1985a-q 1986a; Ben-Hur et a l . , 1985a; Brasseur et ul., 1985; Chan et al., 1986). Because of superior optical properties of PC compared to hematoporphyrin derivative (HpD), as well as their stability and chemical purity, Ben-Hur and Rosen- thal (1985a) suggested PC as photosensitizers in photodynamic therapy (PDT) of cancer (for recent reviews of HpD PDT see Dougherty, 1985; Wilson and Jeeves, 1987). Indeed, PC can photosensitize the destruction of transplanted tumors in exper- imental animals (Selman et ul., 1986).

The photochemistry and photobiology of PC is under study. The action spectrum of AlPC is slightly red-shifted with respect to the absorption spectrum of the free dye (Ben-Hur and Rosenthal, 1986b) and AlPC-sulfonate (AIPCS) can cause photo- hernolysis of red blood cells, indicating the induction of membrane damage (Ben-Hur and Rosenthal, 1986~) . In the presence of molecular oxygen PC can produce superoxide anion radicals (Ben-Hur et a l . , 1985a) and singlet oxygen (Rosenthal et a l . , 1986). However, although O2 is required for PC photosen- sitization neither 0; nor '02 appear to be involved in the induced phototoxicity. Scarce information is

*To whom correspondence should be addressed. tAbbreviations: AIPCS, chloroaluminum phthalocyan-

ine tetrasulfonate; DMEM. Dulbecco's modified Eagle's medium; HpD, hematoporphyrin derivative; PBS, phos- phate buffered saline; PC, phthalocyanine; PDT, photo- dynamic therapy; TCA, trichloroacetic acid.

available concerning the effect of PC photosensitiz- ation on biochemical processes in the cell, aside of the observation that purified enzymes in solution can be inactivated (Spikes and Bommer, 1986).

In the present work, we determined the effect of AlPCS photosensitization on several biochemical pathways of cultured Chinese hamster cells metab- olism. It was found that macromolecules synthesis and uptake of nucleosides and amino acids, as well as glucose oxidation are inhibited by this treatment. The sensitivity of the latter process was correlated with phototoxicity and is consistent with the obser- vation of mitochondria1 damage, photosensitized by AlPCS in human lymphocytes.

MATERIALS AND METHODS

Cell culture. Chinese hamster cells, line V79-B310H, were grown as a monolayer in 50 mm plastic Petri dishes using Dulbecco's modified Eagle's medium (DMEM) con- taining 10% fetal calf serum. The cells doubled in number every 8-9 h at 37°C in a humidifed atmosphere containing 5% CO,. Survival of cells was determined using colony- forming ability as an endpoint. To determine survival in log-phase, cells (2 x 10' per dish) were plated and treated after an overnight growth. After treatment the cells were suspended by trypsinization, diluted and plated to obtain ca. 100 colonies per dish. Colonies were stained with methylene blue and counted after 7-8 days of growth. To determine survival in plateau-phase, cells were treated 3 days after plating (2 X 10' cells per dish). At the time of treatment the cells formed a confluent monolayer and their number was ca. 7 x 1oh per dish. After treatment the cells were processed as for log-phase. Plating efficiency was over 80% in log-phase and ca. 60% in plateau-phase. Unlike HpD treated cells (Christensen et al . , 1985), AlPCS photosensitization did not inhibit the detachment of cells from substratum by trypsin.

65 1

652 E. BEN-HUR ei ul.

Chemicals. All biochemicals were of the highest purity available commercially. AIPCS was synthesized and pur- ified by cstablished methods (Moser and Thomas, 1983). The condensation of 4-sulfophthalic acid gave the tetrasul- fonated product which had the characteristic absorption of a PC (Am,,, = 674 nm, E = 1 x lo5 e mol--' cm-I) and eluted as a single peak on HPLC. The dye was stored as a 350 pM stock solution in phosphate buffered saline (PBS), filter sterilized and added to the growth medium overlaying the cells to obtain the final concentration. ['HIThyrnidine (40.5 Ci rnrnol-I), ['Hluridine (17.4 Ci mmol- ' ) and ['H]Ieucine (38.8 Ci mmol-I) were obtained from Nuclear Research Center-Negev, Isarel. D- [lJC(U)]glucose (257.7 mCi mmol-') was from New England Nuclear, Boston, MA.

Biochemical assays. DNA, RNA and protein syntheses were measured as incorporation of ['Hlthymidine, [3H]uri- dine and [ZH]leucine, respectively, into acid-insoluble frac- tion. Radioactive precursors were added into DMEM at 5 pCi me-l and incubated with log-phase cells for 1 h. Incorporation was terminated by rinsing the cell mono- layer three times with cold PBS, followed by the addition of cold 5% TCA. After 30 min in the cold, the TCA was removed, the plates were rinsed with ethanol and air dried. Macromolecules were dissolved by adding 0.5 me hydroxide of hyamine and incubating overnight in the cold. The solution was then transferred into 10 me toluene- based fluor, and radioactivity counted in a liquid scintil- lation spectrometer (Packard). Incorporation of radio- activity was calculated on a per-cell basis and the results are presented as a percent of untreated control. Triplicate plates were used for each datum point. Standard errors were less than 10% and are not shown in the figures for clarity.

To determine the uptake into the acid-soluble fraction of the cells, 1 me of the TCA solution was transferred into 10 mC water-miscible fluor (Insta-Gel) after 30 min extraction of the cells, and radioactivity counted as above.

Glucose oxidation was measured as the release of I4CO2 from ['4C]glucose, as described previously (Ben-Hur ei ul., 1985b). Determinations were made in duplicates.

Ulirustruciurul studies. Peripheral human lymphocytes were obtained from healthy donors, separated and treated with AIPCS, as described by Kol ei ul. (1986). Following treatment, the cells were fixed in 1% glutaraldehyde in 0. I M sodium cacodylate buffer (pH 7.2) and postfixed in 1 % osmium tetroxide. Dehydration was performed in a graded series of ethanol and embedding in Araldite 502. Thin sections were stained with uranyl acetate and lead citrate and studied under Philips 201C Electron Micro- scope.

Light exposure. Prior to light exposure the growth medium was removed and 3 me PBS was added into each plate. The dishes were then exposed at room temperature to white light from a bank of three 40 W 'cool white' fluorescent tubular lamps (Sylvania) held in a reflector. The light fluence rate at the level of the cell monolayer was 55 W r r 2 . About 15% of the light emitted was in the red (60G-700 nm).

RESULTS

Chinese hamster cells photosensitized in log- phase by AlPCS display a survival curve charactized by a large shoulder followed by a steep exponential decline (Ben-Hur and Rosenthal, 1986a). Figure 1 shows that cells in plateau-phase are about twice as sensitive to AlPCS photosensitization as are cells in log-phase. Following a light fluence that kills most of the cells in log-phase there is some inhibition of DNA and RNA synthesis immediately after

I L 20 40 , \ , 60 80

4 uM APPCS, 2 4 hr

LlGHT FLUENCE ( k J / m 2 )

Figure 1. Survival of Chinese hamster cells photosensi- tized by AIPCS. Cells in log-phase or plateau-phase, as indicated, were incubated for 24 h with 4 FM AIPCS and then exposed to white light. Survival was determined from colony-forming-ability, as described in Materials and

Methods. N is the average cellular multiplicity.

4uM APPCS, Id hr 66 KJlm'

4uM APPCS, Id hr 80 - 66 KJlm'

I

0 I I I I 1 2 4

T IME AFTER I LLUMINATION h

Figure 2. Inhibition of D N A and RNA syntheses in Chinese hamster cells photosensitized by AIPCS. Cells in log-phase were incubated for 18 h with 4 p M AlPCS and then exposed to 66 kJ m-z of white light. Incorporation of ['Hlthymidine and ['Hluridine, as indicated, into acid- insoluble fraction, was determined at various times after light exposure. The results are shown as percentage of control, untreated cells. Open circles are for cells exposed

to light in the absence of dye.

exposure as reflected by inhibition of incorporation of thymidine and uridine respectively, into acid- insoluble fraction (Fig. 2). This inhibition becomes more pronounced with time, reaching a maximum 3 h after exposure. Both DNA and RNA syntheses are inhibited to about the same extent. The fluence

Phthalocyanine photosensitization 653

80

* L

0 Y

.., 0

0

- - ? 60- 5

40-

20

response of DNA, RNA and protein syntheses, when measured 3 h after exposure to obtain a maxi- mal effect, is shown in Fig. 3. The inhibition curves are similar in shape to the survival curve, with a shoulder region followed by a sharp decline. Although protein synthesis appears to be more sen- sitive to photosensitized inhibition, the differences were not statistically significant. Neither light alone nor AlPCS in the dark inhibited macromolecular syntheses at the dose range studied in this work.

- ;:I 4 " ~ A ~ P C S . 18 hr-40 K J / ~ '

V79 - B310H

4uM AlPCS. 18 h r

3 h r after l i g h t

I 1 20 4 0 KO

L l M T FLUENCE ( U d )

Figure 3. Inhibition of DNA, RNA and protein syntheses in Chinese hamster cells photosensitized by AIPCS. Cells in log-phase were incubated for 18 h with 4 kA4 AIPCS and then exposed to white light. Incorporation of ['Hlthy- midine (W), [3H]uridine (A) and ['Hlleucine (0) into acid-insoluble fraction was determined 3 h after light

exposure.

The apparent inhibition of incorporation of radio- active precursors into the macromolecules in the cell could be due, at least in part, to inhibition of their uptake into the cellular pool from the growth medium. The results in Table 1 show that some inhibition of uptake into acid-soluble fraction does occur, but it is not sufficient to explain all the inhibition of incorporation into acid-insoluble fraction.

Figure 4 shows the effect of AlPCS photosensitiz- ation on glucose oxidation in log-phase cells. A light fluence that kills about half the cells caused only a small inhibition of glucose oxidation when measured immediately after exposure. Inhibition increased progressively with time, reaching a maximum after 3 h. At a later time the cells recovered slowly but not completely even after 24 h. After a high fluence (66 kJ m-*) that kills most of the cells inhibition was pronounced immediately after exposure (62%),

0 - 10 - 0

Figure 4. Inhibition of glucose oxidation in Chinese ham- ster cells photosensitized by AIPCS. Cells in log-phase were incubated for 18 h with 4 AlPCS (0) or in the absence of dye (0) and then exposed to 40 kJ m-, of white light. Glucose oxidation in 1 x loh cells was determined at various times after light exposure, as described in Materials and Methods. The results are shown as percent- age of control, untreated cells, which released -2000 cpm of '"CO,. Error bars are shown when larger than the data

points.

increased to 90% after 3 h and no recovery was observed. The fluence-response curves for AlPCS photosensitized inhibition of glucose oxidation 1 and 3 h after exposure are shown in Fig. 5 . In the fluence range studied the response appears to be exponential throughout. However, the presence of a shoulder at fluences lower than those used is not ruled out. It is noteworthy that inhibition of glucose

Table 1. Effect of AIPCS photosensitization on ['Hlthymidine and ['Hlleucine incorporation into acid-soluble and acid-insoluble fractions of Chinese hamster cells

Light fluence

(kJ m-*)

[ 'HlThymidine [ 'HILeucine

Acid-soluble Acid-insoluble Acid-soluble Acid-insoluble -~ ~

( Y O ) ( O% 1 ( "ILI) ("/.I

0 50 66 82

100 f 6.2 100 2 5.0 100 t 4.9 100 k 3.1 49 t 0.5 39 t 4.1 75 t 3.2 51 ? 8.7 45 ? 3.2 26 ? 2.3 66 t 1.5 27 t 0.3 41 t 5.1 15 t 1.6 64 t 8.1 11 2 1.8

Chinese hamster cells in log-phase were incubated for 18 h with 4 k M AIPCS and then exposed to white light. After 3 h at 37°C ['Hlthymidine or ['Hlleucine were added for 1 h and incorporation into acid-soluble and acid-insoluble fractions of the cells was determined as described in Materials and Methods.

E. BEN-HUR el al.

L I u H T FLULNCL l K J / &

Figure S. Inhibition of glucose oxidation in Chinese ham- ster cells photosensitized by AIPCS. Cells in log-phase ( 0 . A ) or plateau-phase (.,A) were incubated for 18 h with 4 p M AIPCS and then exposed to white light. Glucose oxidation in 1 x loh cells was determined at 0 h (0,O) or 3 h ( A , A ) after light exposure. For details, see legend to

Fig. 4.

oxidation occurred in log-phase cells after light flu- ences about twice as large as in plateau-phase cells. To directly observe the subcellular targets for AIPCS photosensitization electron micrographs of human lymphocytes were prepared (Fig. 6). The light fluence used killed about SO% of the cells (Kol et al . , 1986). Evidently, 6 h after treatment the most prominent changes are in the mitochondria and to some extent in the nucleus.

DISCUSSION

The results obtained for inhibition of macro- molecules synthesis by AIPCS photosensitization differ from those previously described for H p D photosensitization (Moan et af., 1983). Thus, while DNA synthesis is by far the most sensitive to H p D and light, it seems to be as sensitive to AIPCS and light as RNA and protein syntheses (Fig. 3). In addition, inhibition of DNA synthesis is maximal immediately after H p D photosensitization and dis- appears during further incubation of the cells (Moan et af., 1983). After AIPCS photosensitization immediate inhibition is slight and it becomes more pronounced with time (Fig. 2). In both HpD and AIPCS photosensitized cells there is some inhibition of nucleosides and amino acids uptake (Moan et al. , 1983; Table 1) which, however, is lower than inhibition of incorporation into the acid-insoluble

fraction, indicating some real inhibition of macro- molecular syntheses. However, reduced incorpor- ation could also result from increased intracellular dilution of the radioactive precursor or its metab- olites due to expanded pools, e.g. thymidine tri- phosphate, after photosensitization.

Inhibition of glucose oxidation by AIPCS and light is significantly more pronounced than inhib- ition of macromolecular syntheses, particularly in the lower fluence range (compare Figs. 3 and 5 ) . Glucose oxidation reflects the activity of various enzymes involved mostly in the tricarboxylic acids (citric acid) cycle. Inhibition of some of them could contribute to the observed effect. However, effect on enzymes participating in the beginning of the cycle (e.g. aconitate hydratase) would be more effective in inhibiting glucose oxidation than on those at its end (e.g. malate dehydrogenase). The high sensitivity of glucose oxidation and the obser- vation that it is correlated with cell killing (compare Figs. 1 and 5 ) make it an attractive candidate as an important target for cell inactivation. This is further supported by the observation that mitochondria1 damage is conspicuous in electron micrographs of AIPCS photosensitized human lymphocytes (Fig. 6) and that PC localize selectively in mitochondria (our unpublished results). If this interpretation is correct then inhibition of macromolecular syntheses could be a secondary result caused by depletion of energy resources of the cell. Whether this is indeed the case should be resolved by experiments that will determine the effect on individual enzymes. It is noteworthy that the initial changes observed after HpD PDT are in the mitochondria (Kato et al., 1986), which appear to be a major target for this treatment (Wilson and Jeeves, 1987).

Finally, the large difference in sensitivity between log-phase and plateau-phase cells is noted. The possibility that cells in G , phase, a cell-cycle phase in which cells tend to accumulate when reaching confluency, are more sensitive to AIPCS photosensi- tization than cells in other phases is unlikely, since it was shown that the response of Chinese hamster cells to AIPCS photosensitization does not change significantly throughout the cell-cycle (Ben-Hur and Rosenthal, 198Sb). Another possible explanation is related to the lower p H of the growth medium in plateau-phase, which might enhance the uptake of AIPCS into the cells, leading to enhanced photosen- sitivity. Indeed, lower pH was found to enhance photosensitivity of Chinese hamster cells to AlPC (Ben-Hur and Rosenthal, 1985c) and human cancer cells to H p D (Moan et af., 1980). In experiments in which the uptake of AIPCS was measured after 24 h incubation with log-phase or plateau-phase cells, it was found that the latter contained almost twice as much dye (data not shown). This finding supports enhanced uptake of AIPCS as the basis of enhanced photosensitivity in plateau-phase.

h

v D

h

v m

Phthalocyanine photosensitization 655

656 E. BEN-HUR et al.

Acknowledgements-We thank Mrs. M. Minzberg for technical assistance.

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