Photochemistry and Photobiology Vol. 54, No. 2, pp. 163-166, 1991 Printed in Great Britain. All rights reserved.

003 1-8655191 $03.00+0.00 Copyright 0 1991 Pergamon Press plc

RAPID COMMUNICATION

PTHALOCYANINE-INDUCED PHOTODYNAMIC CHANGES OF CYTOPLASMIC

FREE CALCIUM IN CHINESE HAMSTER CELLS

E. Ben-Hur*, T.M.A.R. Dubbelman and J. Van Steveninck

Sylvius Laboratory, Dept. of Medical Biochemistry P.O.Box 9503, 2300 RA Leiden, The Netherlands

(Received 1 April 1991; accepted 10 May 1991)

Abstract-- Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca”],, from about 0.2 pM to 1 p M within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca”] , . These findings raise the possibility that changes in [Ca“], after photodynamic treatment may be relevant to cell killing a n d / o r o t h e r b i o l o g i c a l r e s p o n s e s o f the cells, e.g. release ofeicosanoids.

INTRODUCTION

Phthalocyanines (Pc)’ are efficient photosensitizers of mammalian cells (Ben-Hur and Rosenthal, 1985a; Ben-Hur and Rosenthal, 1985b; Brasseur et a l . , 1985). Pc are currently being studied as second-generation sensitizers for PDT because of their improved properties for that purpose compared to HPD (for recent reviews see Van Lier, 1990; Ben-Hur, 1991).

The molecular mechanism by which PDT, using either HPD or Pc, causes cell killing and other biological effects is still obscure. The fast release from cultured cells of eicosanoids after HPD-PDT (Henderson and Donovan, 1989) and of clotting factor after Pc-PDT (Ben-Hur et al., 1988) could be triggered by [Ca”], increase. Very fast change in [Ca”], is a trigger for many cell responses (Rasmussen, 1986) and could also be involved in cell killing (Nicotera et al., 1990; Farber, 1990). To our knowledge [Ca”]. changes following PDT have not been reported. In this report we describe fast changes in [Ca”], in Chinese hamster cells photosensitized with AlPcC1. The observation that [Ca”], increased together with cell killing suggest that the two processes may be causally related.

MATERIALS AND METHODS

Chemicals. All chemicals were analytical grade. AlPcCl was obtained from Eastman Kodak (Rochester, NY) and was stored as a stock solution of 1 mM in dimethylformamide at room temperature. Fura-2/AM was from Sigma (St. Louis, MO) and stored as 2 m M stock solution in dimethylsulfoxide at -2O’C.

Cell culture. CHO epithelial cells, clone KI, ATCC number CCL61, were grown attached to plastic petri dishes in Ham’s F10 medium supplemented with 15% newborn calf serum in a humidified atmosphere containing 5% CO, at 37OC.

Cell survival. Cells (200-2000) were plated in 5 cm plastic dishes. After 4 h AlPcCl was added at 1 pMand incubation continued for 2 h. The medium was then replaced with DPBS and the cells exposed to light. Growth medium was added and incubation continued for 7 d. Colonies were then stained and counted. Triplicate plates were used for each datum point. To test for the effect of fluoride, the DPBS contained 5 mMNaF during illumination. Standard errors were < 10%.

Light exposure. The light source was a slide projector equipped with a 150 W quartz halogen light bulb. The light was filtered by a cut-off filter ( A > 605 nm). The incident fhence rate was ZOO w m-’.

Measurement of cytosolic f r e e Ca2+. [Ca2’], was measured as described previously (Malgaroli et a ] . , 1987), with some modifications. Briefly, fura-Z/AM was loaded into the cells at room temperature by addition of the dye into growth medium at 2 pMfor 30 min before light exposure.

’;To whom correspondence should be addressed. +Abbreviations: AlPcC1, chloroaluminum phthaiocyanine; AlPcS, sulfonated aluminum phthalocyanine; CuPcS, sulfonated copper phthalocyanine; [Ca2+], , cytosolic free Ca2+; DPBS, Dulbecco‘s phosphate buffered saline; HPD, hematoporphyrin derivative; pc, phthalocyanine; PDT, photodynamic therapy.

163

164 E. BEN-HUR et al.

Prior to assay, the cells (about 10' in a 5 cm petri dish) were rinsed 3 times with DPBS and suspended in 3 ml DPBS by scrapingwith a rubber policeman. Fluorescence of the cell suspensionwas then measured in an Aminco SPF 500 spectrofluorimeter at two excitation wavelengths, 340 nm and 385 nm, using A e m = 505 nm. The ratio F3.,,JFJB5 was used to calculate [Ca"], as described by Grynkiewicz et al. (1985). Variation between duplicates in the same experiment was < 3%. Because differences between experiments were 30-40%, the results shown are of typical experiments.

RESULTS

as a function of time after AlPcCl-PDT in CHO cells. Evidently, there is a rapid increase in lCa2+], immediately after light exposure, reaching a maximum in about 5 min. Thereafter, [CaZt], declines with a half-time of about 30 min. Table 1 shows the dose-response of [Ca''], changes, measured 5 min after light exposure. Maximal response is obtained after 40 s illumination (8 kJ m-2). No change in [Ca2+], occurred after the same light exposure of cells not loaded with AlPcCl. Also shown in Table 1 is the effect of light exposure in the presence of fluoride. Previously F- was shown to protect human erythrocytes against photohemolysis sensitized with AlPcS (Ben-Hur et al., 1991). Clearly, F- also protects CHO cells against phototoxicity induced by AlPcCl. Concomitantly, changes in [Ca2'], are also reduced by fluoride (Table 1).

Figure 1 shows the changes in [Ca2+]

1

1

0 10 20 30 40 50 60 70

Fig. 1. Kinetics of [Ca2'], changes in CHO cells. Cells were incubated with 1 pMAlPcCl for 2 h and with 2 pMfura-L/AM for 30 min and then exposed to 60 s red light (0) or kept in the dark ( 0 ) . [CaZtli was measured at various times after light exposure.

Table 1. [CaZt], changes and cell survival

h9 ( S )

0 10 20 30 40 60

[ca2+] ( X ) Control 5 mMNaF 100 100 225 151 264 165 278 186 298 199 296 21 4

Cell survival (X) Control 5 mM NaF 100 100 78 85 32 77 5.8 71 0.5 65

< 0.01 52

DISCUSSION

The involvement of Ca2+ in the regulation of a large number of physiological processes in mammalian cells is well established. More recently it became apparent that Ca2+ can also play a determinant role in a variety of pathological and toxicological processes (Nicotera etal., 1990,

Rapid Communication 165

Farber, 1990). is usually maintained in the cell at about 100 nM against an outside concentration of hr 1 mM. A disruption of intracellular Ca2+ homeostasis is frequently associated with the early development of cell injury (Jewel1 et al., 1982). This led to the hypothesis that [Ca"], increase may be a common step in the development of cytotoxicity (Nicotera et a]., 1990). Our results that [Ca2+], increases immediately after AlPcC1-PDT in the biological dose range are consistent with this hypothesis, and raise the possibility that [Ca"], changes play a role in PDT-induced cell killing. The protection by F- against both cytotoxicity and increased [Ca"] lends further support for this idea, although it does not constitute a proof. Further experiments are required to establish a causal relationship between [CaZt], changes and PDT cytotoxicity, as well as the generality of the phenomenon for other photosensitizers and cell lines.

[Ca*+], increases dramatically in a very short time as a result of various stimuli in a transient fashion, similar to that observed after PDT (Fig. 1). For example, Ca2+ mobilizing hormones, such as vasopressin, produce a transient elevation of [Cazt]l in hepatocytes to 0.4-1 pM, followed by a slow return to basal levels (Charest et al., 1983). HPD-PDT is known to cause release of eicosanoids such as thromboxane A, (Fingar et al., 1990) and prostaglandin E, (Henderson and Donovan, 1989). Clotting factors are also released from endothelial cells as a result of AlPcS-PDT (Ben-Hur et al., 1988). The mechanism underlying the above release is not known but elevated [Ca2+], is likely to be involved. This is supported by the observation that AlPcS-PDT induces amylase release from exocrine pancreatic cells but not from a pancreatic carcinoma cell line (Matthews and Cui, 1990). Elevation of [Ca2'], by the ionophore A23187 elicited secretion of amylase from the normal pancreatic cells but not from the cancer cells. However, direct measurements of [Ca"], were not done in those pancreatic cells after PDT and the role of changes in free cytoplasmic Ca2+ in the release process remains to be determined.

Elevated [Ca"], is involved in trans-membrane signal transduction for transcriptionally- controlled enzyme induction. HPD-PDT was recently shown to induce transcription and translation of the oxidative stress-related enzyme heme oxygenase (Gomer etal., 1991). Atransient elevation of [Ca"], could be an early signal for this response.

elevation is not known. In principle it could involve enhanced Cazt influx and/or release of Cab' stored in endoplasmic reticulum. It is noteworthy in this connection that CuPcS induces rapid Ca2+ release in vitro from sarcoplasmic reticulum in the dark (Abramson et al., 1988) and that oxidation of protein sulfhydryl groups by toxic agents inhibits ATP-dependent Ca" sequestration by rat liver microsomes (Thor et a]., 1985). It remains to be seen whether AlPcC1-PDT acts by a similar mechanism.

In conclusion, a transient elevation of [Ca2'], in CHO cells after AlPcC1-PDT has been demonstrated. This increase occurs in the biological dose range and could be involved with the release of various substances induced by PDT.

Acknowledgement. This work was supported by a grant from the Netherlands Cancer Foundation (IKW

[Ca"],

The mechanism by which AlPcCl-PDT induces [Ca2+]

89-01 1.

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