rapid initiation of apoptosis by photodynamic therapy

Photochemistry and Photobiology, 1996, 63(4): 528-534

Rapid Initiation of Apoptosis by Photodynamic Therapy

Yu Luol, C. K. Chang2 and David Kessel*I ’Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit, MI, USA and 2Department of Chemistry, Michigan State University, East Lansing, MI, USA

Received 17 July 1995; accepted 2 January 1996

ABSTRACT

Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD,,), measured by the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer an- tagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucleosomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD,, PDT doses. Photodamage from two cationic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD,,, LD,, or LD9, PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may there- fore be a major determinant of the initiation and rate of progression of apoptosis.

INTRODUCTION

photodynamic therapy (PDT)? has been demonstrated both in vitro (2 ,3) and in vivo (4). The rapid DNA cleavage to nucleosome oligomers after PDT (sometimes within 10-30 min) suggests the initiation of a late step in the apoptotic process. In a series of experiments involving murine leukemia L5 178Y cells and an aluminum phthalocyanine, apoptosis was associated with rapid phospholipase C activation and a resulting release of Ca2+ (5,6). An increased influx of Ca2+ is also an early event in the photodynamic action of many photosensitizing agents (7,8).

The initiation of apoptosis after PDT appears to be a function of both sensitizer and cell line. With Photofrin, PDT- induced apoptosis was observed in two of three malignant cell lines examined (2). Photodynamic therapy with merocyanine 540 induced apoptosis in one leukemic cell line but not in two others (9). It should be pointed out that DNA “ladder” formation was often used as the sole criterion for apoptosis. Although the appearance of ladders, i.e. fragmentation to the nucleosome level, is often considered to be sufficient for the demonstration of apoptosis, internucleosomal cleavage is not uniformly associated with apoptotic cell death (10-12).

In experiments carried out with murine leukemia L5178Y cells, PDT with aluminum phthalocyanine resulted in apoptosis and DNA ladder formation, even when no viable cells survived. In contrast, PDT with two silicon phthalocyanines resulted in death by apoptosis, with DNA ladder formation detected at PDT doses that reduce the viability of a cell population to 90% of control (LD,,). At still higher PDT doses, DNA fragmentation did not proceed beyond the 50 kb size (13). This result was interpreted to indicate inhibi- tion, at a high PDT dose. of endonucleases or endonuclease activators. We carried out a similar study with a porphycene dimer (PcD) and reported that a high drug dose in the dark could initiate internucleosomal DNA cleavage, but subsequent irradiation abolished this effect (14). The present study was begun to provide further information on the relationship

Apoptosis is a mode of cell death initiated by cellular signals and resulting in a specific pattern of DNA fragmentation and conversion of cells to apoptotic bodies ( 1 ) . Apoptosis after

*To whom correspondence should be addressed at: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI 48201, USA. Fax: 313-577-6739; e-mail: [email protected].

0 1996 American Society for Photobiology OO31-8655/96 $S.OO+O.OO

tAbbrwiations: AO, acridine orange; DCKC, dicationic ketochlorin; FHS, Fischer’s medium (10% horse serum) buffered with 20 mM HEPES instead of NaHCO,; H0342, Hochst dye 33342; LD,, conditions that reduce the viability of a cell population to x% of control ( M l T assay); MCP, monocationic porphyrin; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PcD, porphycene dimer; PDT, photodynamic therapy; R123, rhodamine 123; SnET2, tin etiopurpurin; TDPH, trimethylaminodiphenylhex- atriene; trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

528

Photochemistry and Photobiology, 1996, 63(4) 529

' A '

C

0 C

B

D Figure 1. Photosensitizing agents used in this study. A: Tin etiopurpurin (SnET2). B: Dicationic chlorin (DCKC). C: Monocationic porphyrin (MCP). D: Porphycene dimer (PcD).

between photodamage and apoptosis and to examine the pos- sibility that certain types of photodamage could delay or abolish the rapid onset of apoptosis. We tested this hypothesis in a study involving four sensitizers with two different sites of photodynamic action.

MATERIALS AND METHODS Chemicals. Tin etiopurpurin (SnET2) was provided by Dr. Alan Morgan (PDT Pharmaceuticals, Hollister, CA). The synthesis of PcD (15) and of monocationic porphyrin (MCP) (16) have been reported. Preparation of dicationic ketochlorin (DCKC) is described in the Appendix. Structures of all four sensitizers are shown in Fig. 1 . Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Stock solutions were prepared in degassed 10 mM NaOH, stored at 4°C for <1 week and discarded if any color was evident. Trimethylami- nodiphenylhexatriene (TDPH) and other fluorescent probes were obtained from Molecular Probes (Eugene, OR).

Cell lines. The P388 cells were grown in Fischer's medium (GIB- CO-BRL, Grand Island, NY) supplemented with 10% horse serum, 0.1 mM glutamine, 0.1 mM mercaptoethanol and gentamicin. Cells were used in the log phase of growth. All incubations were carried out in a HEPES-buffered formulation of Fischer's medium termed FHS.

PDT experiments. Cell suspensions in FHS were incubated with specified levels of the different photosensitizers for 15 min at 37°C. Cell pellets were then collected by centrifugation, taken up in cold FHS and irradiated at 10°C. In some studies, 3 m M trolox was present during loading incubations and irradiation to antagonize effects of activated oxygen species (17). The light source was a 600 W quartz-halogen lamp with IR radiation attenuated by a 700 nm cut-off filter and a 10 cm layer of water; the bandwidth was further delineated by a 600 nm cut-off filter. The total power output at 600-700 nm, measured with an EGG model 450 photometer, was 5 mW cm (light doses = 1.5 J cm ,). The cell density during such incubations was <2 mg/mL wet weight (approx. lo6 cells/ mL), to prevent the spontaneous initiation of apoptosis that occurs at high cell densities. After irradiation, cells were resuspended in Fischer's medium for specified intervals at 37"C, at a density of 3 X lo5 cells/mL, then collected for gel electrophoresis and fluorescence microscopic examination. To test for viability, aliquots of control and irradiated cells were placed in a CO, incubator for 3 days (initial density = 4 X lo4 cells/mL). Viability was then assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which yields results closely correlated with clonogenic assays (18).

Electrophoretic detection of endonucleosomal DNA cleavage. We examined cells that were (a) untreated, (b) exposed to sensitizers in

the dark and (c) loaded with sensitizers and irradiated. Pellets were collected (wet weight = 5-7 mg) by centrifugation and resuspended in 0.5 mL of buffer containing 150 mM NaCI, 15 mM sodium citrate, 10 mM EDTA and 25 pg of sarkosyl. After thorough mixing, 50 pL of proteinase K (1 mg/mL) w dded and the mixture incubated at 50°C for 2 h. The mixture was cooled to 0°C and 1 mL of ethanol (at -20°C) was added to precipitate DNA, which was collected by centrifugation (12000 g, 20 min), dried in air and dissolved in SO pL of 10 mM Tris, 1 mM EDTA at pH 8 (TE buffer). The DNA levels were quantitated by observing optical density at 260 and 280 nm; in all studies the ratio (A26dA,8J was >1.5. Each DNA preparation (30 pL = approx. 10 pg) was mixed with 1 pL of RNase (10 mglmL) and 6 pL of loading buffer (30% glycerol, 0.1% brom- phenol blue) and incubated for 15 min at 37°C. Samples were then loaded onto 1.5% agarose gels and electrophoresed for approx. 3.5 h (2 V/cm gradient). A series of markers (154-216s bp) was obtained from Boehringerhfannheim (molecular weight marker kit VI). The gels were stained with etbidium bromide, and the DNA bands were visualized under 312 nnl light.

Pulsed-jield gel electrophoresis. After irradiation and additional incubation as described above, P388 cells were washed with phos- phate-buffered saline at 4°C. Cell densities were assessed with a Coulter ZM particle counter to insure that equal numbers were loaded onto gels. Cell samples were then resuspended in L buffer (10 mM Tris, 100 mM EDTA, 20 mM NaCI, pH 7.6). An equal volume of 1% low melting-point agarose in L buffer was added with gentle mixing. Plugs were formed at 0°C and transferred to 1 mL of L buffer containing 1% sarkosyl + 0.5 mg/mL proteinase K. After incubation for 24 h at 50°C the plugs were washed three times in TE buffer (1 h each). Pulsed-field electrophoresis was carried out with a Field Inversion System (BioRad Laboratories, Melville, NY) using 1 % agarose gels in TBE buffer (3.6 mM Tris pH 8, 3.6 mM boric acid and 0.08 mM EDTA) at 4°C. The switch time was linearly varied from 0.5 to 10 s; forward voltage = 180, reverse voltage = 60; time = 20 h. After electrophoresis, the gels were stained with ethidium bromide (0.5 pg/mL) for 45 min and destained in water for 1 h. Fluorescence patterns were acquired using a Panasonic CCD camera (VW BP-310), processed through a Dage MTI 200 digital signal processor and captured with a Truevision Targa system. A broad-band 600 nm filter (bandpass = SO nm) was placed in front of the camera lens to eliminate the infrared radiation from UV lamps.

Fluorescence microscopy. After incubation of P388 cells with LD,, levels of the different photosensitizers, fluorescence microscopy was used to assess sites of sensitizer localization (excitation = 4 0 0 4 5 0 nm, emission = 600-700 nm). To delineate sites of photodamage, control and photodamaged cells were incubated with fluorescent probes immediately after irradiation. Mitochondria1 photodamage was detected with rhodamine 123 (R123), lysosomal photodamage with acridine orange (AO) (19), membrane photodamage with TDPH (20) and DNA fragmentation with the DNA probe Hochst dye 33342 (H0342). For the latter, cells were stained in S pM dye for 5 min at 3 7 T , and numbers of apoptotic nuclei per field of 100 cells were scored. Images were acquired using 350 nm excitation, with emission limited to 4 0 0 4 5 0 nm, using a Dage 68 SIT camera fitted with a digital signal processor (21).

RESULTS Reproducibility of results

Data reported here represent results of experiments that were repeated at least three times. Typical observations from gel electrophoresis and fluorescence microscopy are in Figs. 2-7.

Sites of photodamage

Fluorescence microscopy showed that the cationic agents MCP and DCKC were both localized at the cell membrane (Fig. 2). Fluorescent patches observed on cell surfaces are typical of cell-surface markers and can also be seen with TDPH, an agent that labels only cell surfaces after short incubations (20,21). Using LD,, PDT doses of either sensi-

530 Yu Luo et al.

Figure 2. Sites of localization and photodamage of the cationic photosensitizers as detected by fluorescence. From left: MCP, KCDC, TDPH (a probe for cell surface photodamage), TDPH after MCP photodamage, TDPH after KCDC photodamage.

tizer, we observed no alteration in fluorescence patterns of the lysosomal probe A 0 or the mitochondria1 probe R123 (not shown), but we found a substantial promotion of TDPH migration beyond the outer cell surface (Fig. 2) , indicative of membrane photodamage (21). These results show that the cationic sensitizers very selectively mediate photodamage only at membrane loci. Photodynamic therapy with SnET2 (21) or PcD (14) is known to cause lysosomal photodamage.

Electrophoretic evidence of apoptosis

Figure 3 (lane 4) shows the typical DNA ladder fragmentation pattern observed upon irradiation of cells previously incubated with S p M PcD (LD,, conditions). This pattern was detected when cells were incubated for at least 30 min at 37OC after PDT; in results shown here, a 60 min incubation time was used. This degradation pattern was not ob-

1 2 L 3 4 5 6

Figure 3. Endonucleosomal cleavage 60 min after PDT. Lane 1 = markers, lane 2 = control, lane 3 = 5 p M PcD (dark), lane 4 = 5 p M PcD + light, lane 5 = 30 p M PcD (dark), lane 6 = 30 pM PcD + light.

served when the incubation was carried out at 0°C. When the PcD concentration was increased to 30 pM, we observed DNA ladder formation in the absence of light (lane S), but subsequent irradiation abolished this effect (lane 6). The latter PDT dose yields a >99% decrease in cell viability. Sim- ilar results were obtained with SnET2 (not shown), including the high-dose dark toxicity. In all cases, relative numbers of apoptotic nuclei were assessed by fluorescence microscopy. Chromatin fragmentation was undetectable in cells used in lanes 2, 3 and 6, 31 t 8% in lane 4; 40 t 6% in lane 5 .

When cells were loaded with MCP and then irradiated (LD,, conditions), we observed no DNA ladder formation 1 4 h after PDT. Results shown in Fig. 4 were obtained 1 h after PDT, using 0.3 (lane 3), 1 (lane 4) or 3 pM MCP (lane 5 ) , representing approx. LDIo, LD,, and LD,, PDT doses. Photodynamic therapy with PcD resulted in DNA ladder formation (lane 6), but the presence of MCP inhibited the rapid endonucleosomal degradation of DNA induced by PcD. Cells loaded with PcD (5 pM) + MCP (0.3 pM, lane 7; 1 pM, lane 8 or 3 pkl, lane 9) and irradiated showed no evidence of endonucleosomal DNA fragmentation after 1 h. Examination by fluorescence microscopy revealed <3% apoptotic cells in preparations used in all lanes except for lane 6 (37 5 5%).

1 2 3 4 5 6 7 8 9

Figure 4. Endonucleosomal cleavage 60 min after PDT. Lane 1 = markers, lane 2 = control P388 cells, lane 3 = 0.3 p M MCP, lane 4 = 1 p M MCP, lane 5 = 3 pJ4 MCP, lanes 6-9 = 5 pM PcD + varying levels of MCP, lane 6 = none, lane 7 = 0.3 p M MCP, lane 8 = 1 pM, lane 9 = 3 p M . All cell suspensions were irradiated except for the control (lane 2).


1 2 3 4 5 6 7 8 9

300

50

Figure 5. Effects of sensitizers and combinations on endonucleo- soma1 cleavage 60 min after PDT. Lane 1 = markers, lane 2 = P388 unincubated control, lane 3 = P388 incubated control, lane 4 = SnET2 (3 pM) + light, lane 5 = PcD (5 pA4) + light, lane 6 = SnET2 (3 pM) + PcD (5 p M ) + light, lane 7 = DCKC (3 p M ) + light, lane 8 = DCKC (3 phf) + PcD (5 p h f ) + light, lane 9 = DCKC (3 pM) + SnET2 (3 pM) + light.

We also examined the effect of SnET2 and DCKC on PcD-induced apoptosis, along with the effect of DCKC alone (Fig. 5). With LD,, PDT doses of SnET2 (lane 4) or PcD (lane 5 ) , a typical apoptotic pattern of DNA degradation was observed. Irradiation of cells loaded with both SnET2 and PcD also yielded this pattern (lane 6). We did not detect DNA ladders with LD,, PDT doses of DCKC (lane 7), DCKC i SnET2 (lane 8) or DCKC + PcD (lane 9). All of these studies involved I h incubations at 37°C after PDT. Relative numbers of apoptotic cells, indicated by fluorescence microscopy, were <3% in lanes 2, 7-9; 8 5 3% in lane 3, 24 1 5% in lane 4, 29 t 3% in lane 5 , 34 -t- 5% in lane 6.

Pulsed-field gel electrophoresis was used to examine early stages of chromatin fragmentation (Fig. 6). Some 50 kb DNA was found in control cells (lane 2); the level was not significantly different in dark controls (lane 3 ) . Exposure of cells to 3 yM SnET2 or 5 pM PcD + light (LD,, conditions), or to 30 yM PcD or SnET2 in the dark, did not significantly increase the amount of 50 kb DNA (lanes 4 and 5) . These results reflect a rapid conversion of 50 kb DNA to smaller fragments (shown in Fig. 3); we could readily detect 50 kb DNA after 10 min under these conditions (not shown). In contrast, there was a substantial accumulation of 50 kb DNA fragments 1 h after cells were exposed to 30 yM PcD and then irradiated (lane 6), or 1 h after an LD,, PDT dose with MCP or DCKC (lanes 7, 8). Accumulation of 50 kb DNA was also observed 1 h after PDT using LD,, levels of PcD + MCP (lane 9) or PcD + DCKC (lane lo), then irradiated. Fluorescence microscopy indicated <5% with apoptotic pat-

Figure 6. Photodynamic therapy-induced DNA digestion producing 50 kb fragments 60 min after PDT. The DNA was analyzed via pulsed-field gel electrophoresis. Lane 1 = 50 kb marker, lane 2 =

control, lane 3 = 5 pM PcD (dark), lane 4 = 5 pM PcD (light), lane 5 = 30 p M PcD (dark), lane 6 = 30 pM PCD (light), lane 7 = 1 p M MCP (light), lane 8 = 3 pM DCKC (light), lane 9 = 5 pM PcD + 1 pA4 MCP (light), lane 10 = 5 pM PcD + 1 p M DCKC (light).

terns of chromatin fragmentation in lanes 2, 3 and 6-10, 21 t 5% in lane 4 and 28 t 5% in lane 5.

Effects of the oxygen scavenger trolox were measured to provide additional information on mechanisms of dark vs light-induced toxicity of photosensitizing agents. Results are summarized in Table 1. Both PDT-induced loss of viability and endonucleosomal cleavage of DNA were inhibited by trolox when LD,, levels of PcD or SnET2 were employed. In contrast, the dark toxicity and DNA fragmentation induced by 30 p M levels of PcD or SnET2 were not affected by the addition of trolox (Table 1). These results indicate that activated oxygen species are not involved in the cytotoxic dark effects of these agents. Trolox also inhibited the

Table 1. phototoxic effects of PcD and SnET2*

Effects of trolox, light and sensitizer concentrations on

Evidence of cytotoxicity Concen- tration Apop- (’% viable

Sensitizer (pM) Trolox Light tosist cells)

SnET2 SnET2 SnET2 SnET2 PcD PcD PcD PcD

5 5

30 30

5 5

30 30

*P388 leukemia cells were incubated with specified levels of SnET2 or PcD for 15 min at 37°C. Where specified, 3 mM trolox was present during this incubation and all subsequent treatments. If specified, cells were irradiated with 0.5 J cm at 660 nm (SnET2) or 630 nm (PcD). Loss of cell viability was measured by the MTT assay 3 days later.

t Apoptosis was assessed by gel electrophoresis (DNA ‘‘ladder’’ formation).

532 Yu Luo eta/.

Figure 7. Fluorescence microscopy of P388 cells stained with H0342 60 min after PDT (LD,, doses with each sensitizer). Top left, control; top right, 5 p M PcD; bottom left, 1 p M MCP; bottom right, PcD + MCP.

phototoxic effects of LDsO PDT doses of MCP or DCKC (not shown).

Fluorescence microscopy

Cells were maintained at 37°C for 1 h and after PDT, then stained with HO342 to detect the chromatin condensation and fragmentation typical of apoptosis. In these studies, PcD or SnET2 yielded similar results. Results of typical studies are shown in Fig. 7. Control cells showed blue fluorescent nuclei with a slightly granular texture (top left). One hour after an LDS0 PDT dose of PcD, we observed a cell population containing approx. 30% apoptotic cells (top right). In contrast, an LD,, PDT dose with MCP yielded slightly swol- len nuclei, but no evidence of condensed chromatin or apoptotic bodies (bottom left). The combination of PcD + MCP (an LD,, PDT dose of each) yielded a more diffuse nuclear staining pattern, but no evidence of apoptotic bodies after 1 h (bottom right).$

DISCUSSION Malignant cell proliferation is often associated with a re- duced frequency of apoptosis, and a concomitant loss of response to chemotherapeutic agents, which, in a normal cell population, elicit an apoptotic response (22). In contrast, there is an almost universal response of neoplastic tissues to PDT, if sufficient sensitizer and light are provided (23,24). If the initiation of apoptosis is a common consequence of PDT, either directly or secondary to vascular shut-down, barriers that limit antitumor responses to conventional drug therapy (25,26) are being circumvented. Reports that PDT can initiate a late stage in the apoptotic process (2-6) are consistent with this proposal.

In a previous report ( 1 3), two types of apoptotic response

$We have observed apoptotic morphology in cells photodamaged with MCP or DCKC after 24 h. Whether this represents delayed apoptosis or apoptosis secondary to necrosis is the subject of ongoing investigations.

to PDT were described: with an aluminum phthalocyanine, cells died by apoptosis at all PDT doses tested, whereas with two silicon phthalocyanines, there was no DNA fragmentation beyond the 50 kb stage, at high PDT doses. With regard to the latter, it has been reported that formation of 50 kb particles may be sufficient for the initiation of apoptosis, even though DNA ladder formation, i. e. endonucleosomal cleavage, is not detected (lQ12).

In this study, we demonstrate the rapid initiation of apoptosis, as demonstrated by endonucleosomal cleavage and apoptotic nuclear morphology, after PDT with either of two photosensitizers (PcD and SnET2) that target lysosomal sites. We also examined the effects of two cationic photosensitizers, MCP and DCKC, that localize at cell-membrane loci and mediate cell-surface photodamage. The LDSo PDT doses with either of these sensitizers elicited DNA fragmentation to 50 kb particles but no internucleosomal cleavage. Moreover, photodarnage from either membrane sensitizer inhibited the rapid internucleosomal DNA fragmentation associated with lysosomal photodamage from PcD or SnET2.

Persistent accumulation of 50 kb DNA was observed 1 h after PDT with MCP or DCKC (Fig. 6); with PcD or SnET2, this species was rapidly converted into nucleosomal oligomers. It is not clear whether the photodynamic effect of the cationic sensitizers represents apoptosis in the absence of endonuclease activity or is the result of a necrotic process that yields only 50 kb DNA fragments. The distinction between these processes is clearly an important one and cannot be made solely on the basis of oligonucleosomal fragmentation (27). Ongoing studies are designed to provide information in this regard. High-dose PcD, in the absence of light, also resulted in internucleosomal cleavage (Fig. 3) but only transient 50 kb DNA formation (Fig. 6). But if cells were exposed to high-dose PcD, then irradiated, we observed a substantial level of 50 kb DNA (Fig. 6) but no evidence of endonucleosomal cleavage (Fig. 3). In this regard, PcD yields an effect similar to that observed with the aluminum phthalocyanine described by He and Oleinick (13).

Antitumor effects associated with PDT result from the formation of cytotoxic oxygen species (28,29). As might be expected, PDT-induced apoptosis can be inhibited by the overexpression of bcl-2 (30). The latter is thought to be involved in antioxidant pathways (31), although a recent report (32) argues against this hypothesis. It is not known whether bcl-2 overexpression would affect apoptosis induced by high levels of SnET2 and PcD in the dark. Cytotoxic effects of high levels of SnET2 or PcD in the dark, resulting in fragmentation of DNA to nucleosomes, clearly do not involve activated oxygen species, as indicated by the failure of trolox to inhibit either cell death or DNA fragmentation. This dark- apoptosis initiation may be analogous to the rapid initiation of apoptosis by the detergent Triton X-100, which, like these sensitizers, has amphipathic properties (33).

The ability of PDT to produce a rapid apoptotic response may be an important element of successful PDT. It is per- haps noteworthy that MCP, an agent that fails to yield a prompt apoptotic response, is considerably less efficacious in vivo (1 6) than are the agents that do, i.e. SnET2 and Pho- tofrin. Whether this pattern will be borne out by further studies remains to be determined.


Acknowledgements-This work was supported by grant CA 23378 from the National Cancer Institute, NIH, DHHS. We thank Drs. Nancy Oleinick and Caroline Dive for providing helpful assistance with regard to procedures used for the detection of apoptosis in these \tudies

APPENDIX Preparation of bis- [ N-[2-(trimethylammonio)ethyl] ]-7,12- dioctyl-3,7,13,17-tetramethyl-8-porphinone-2,1 8-dipropan- amide diiodide salt (DCKC). Dimethyl 7,12-dioctyl- 3,7,13,I7-tetramethyl-8-porphinone-2,18-dipropionate (78 mg, 0.1 mmol) was heated in a mixture of formic acid (20 mL) and concentrated HCI (2 mL) on a steam bath for 1 h. Before the solvent was removed in VUCUO, the hydrolyzed diacid was converted to the acid chloride by stirring with oxalyl chloride (0.2 mL, 1.8 mmol) in dry CH,CI, (50 mL) for 2 h at room temperature. The solvent was evaporated, the residue was redissolved in dry CH,Cl, (30 mL), and this solution was added dropwise to N,N-dimethylethylenedi- amine (60 mg, 0.5 mmol) dissolved in CH,C1, (20 mL). After 1 h, the solvent was removed and the diamide product was purified by chromatography on a silica gel column elut- ed with 15% methanol in CH,CI,; yield, 55 mg. NMR results (in CDCl,): 6 -3.11 (2H, s, NH), octyl: r0.65 (3H, t, Me), 0.85 (3H, t, Me), 0.90 (12H, m, CH,), 1.30 (6H, m, CH,), 1.45, 1.69, 2.19 (2H each, q, CH,), 2.70 (2H, t, 12-CH2), 3.90 (2H, t, 7-CH,)], 1.60 (12H, s, NMe,), 1.79 (4H, m, CH,CH,NH), 2.04 (3H, s, 7-Me), 3.07, 3.04 (2H each, q, CH,NH), 3.19, 3.25 (2H each, t, CH,CO,), 3.48, 3.65, 3.66 (3H each, s, 3, 13, 17-Me), 4.24 (2H, t, lS-CH,CH,CO,), 4.40 (2H, t, 2-CH,CH,C02), 9.15, 9.81, 9.89, 9.94 ppm (1H each, s, meso). FAB-MS results: m/e 915.6531 for (M+H)+; C5,H8,N,O3 requires 9 14.6509. UV-visible spectral data (in CH,CI,): A,,, ( E ~ ) 642 nm (35000), 586 (5600), 545 (1 3 000), 507 (9000), 405 (165 000). The ketochlorin diamide (40 mg) was then methylated by stirring in a mixture of CH,Cl, ( 1 0 mL), methanol (1 mL) and methyl iodide ( 1 mL) for 20 h at 35°C. The solvent was removed in vucuo to leave behind the ammonium salt quantitatively, as monitored by TLC. FAB-MS results: m/e 944.6990, 1071.6008; C5,H,,N,03 requires 944.6979 and C5,H8,N803 %I requires 107 1.6025.

REFERENCES

1. Arends, M. J. and A. H. Wyllie (1991) Apoptosis. Mechanisms and role in pathology. Int. Rev. Exp. Pathol. 32, 223-254.

2. Agarwal, M. L., M. E. Clay, E. J. Harvey, H. H. Evans, A. R. Antunez and N. L. Oleinick (1991) Photodynamic therapy induces rapid cell death by apoptosis in L5178Y mouse lymphoma cells. Cancer Rex 51, 5993-5996.

3 . He, X.-Y., R. Sikes, S. Thornsen, L. W. K. Chung and S. L. Jacques (1994) Photodynamic therapy with photofrin I1 induces programmed cell death in carcinoma cell lines. Photochem. Pho- tobiol. 59, 468473.

4. Zaidi, S. L., N. L. Oleinick, M. T. Zaim and H. Mukhtar (1993) Apoptosis during photodynamic therapy-induced ablation of RIF- 1 tumors in C3H mice: electron microscopic, histopatho- logic and biochemical evidence. Photochem. Photobiol. 58, 771-776.

5. Agarwal, M. L., H. E. Larkin, S. I. A. Zaidi, H. Mukhtar and N. L. Oleinick (1993) Phospholipase activation triggers apoptosis in photosensitized mouse lymphoma. Cancer Res. 53, 5897-5902.

6. Oleinick, N. L., M. L. Agarwal, N. A. Berger, S. Berger, M.-F.

Cheng, S. Chatterjee, J. He, M. E. Kenney, H. E. Larkin, H. Mukhtar, B. D. Rihter and S. I. A. Zaidi (1993) Signal trans- duction and metabolic changes during tumor apoptosis follow- ing phthalocyanine-sensitized photodynamic therapy. Proc. SPIE 1881, 252-261.

7. Specht, K. G. and M. A. J. Rodgers (1991) Plasma membrane depolarization and calcium influx during cell injury by photodynamic action. Biochim. Biophys. Acta 1070, 6 0 4 8 .

8. Ben-Hur, E. and T. M. A. R. Dubbelman (1993) Cytoplasmic free calcium changes as a trigger mechanism in the response of cells to photosensitization. Photochem. Photobiol. 58, 890-894.

9. Traul, D. L. and F. Sieber (1994) Induction of apoptosis and necrosis in leukemia and solid tumor cells by merocyanine 540- mediated PDT. Photochem. Photobiol. 59, 70s.

10. Oberhammer, F., J. L. Wilson, C. Dive, I. D. Morris, J. A. Hickman, A. E. Wakeling, P. R. Walker and M. Sikorska (1993) Apoptotic death in epithelial cells: cleavage of DNA to 300 and/ or 50 kb fragments prior to or in the absence of internucleoso- ma1 fragmentation. EMBO J. 12, 1684-3684.

11. Collins, R. J., B. V. Harmon, G. C. Gob6 and J. F. R. Kerr (1992) Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. /nt. J . Radial. Biol. 61, 451- 453.

12. Walter, P. R., L. Kokileva, J. LeBlanc and M. Sikorska (1993) Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15, 1032-1 040.

13. He, J. and N. Oleinick (1995) Cell death mechanisms vary with photodynamic therapy dose and photosensitizer. P roc. SP / E 2372, 92-96.

14. Kessel, D., Y. Luo, K. Woodburn, C. K. Chang and B. W. Henderson (1995) Mechanisms of phototoxicity catalyzed by two porphycenes. Proc. SPIE 2392, 122-127.

15. Chang, C. K., I. Morrison, W. Wu, S.-S. Chen and S.-M. Peng (1995) Synthesis and structure of A',"-bridgcd porphycenes. J. Chem. Commun., 1173-1 174.

16. Kessel, D., K. Woodburn, B. W. Henderson and C. K. Chang (1995) Sites of photodamage in vivo and in vitro by a cationic porphyrin. Photochem. Photobiol. 62, 875-88 I.

17. Forrest, V. J., Y.-H. Kang, D. E. McLain, D. H. Robinson and N. Ramakrishnan (1 994) Oxidative stress-induced apoptosis prevented by trolox. Free Rudical Bid. & Med. 16, 675-684.

18. McHale, A. P. and L. McHale (1988) Use of a tetrazolium-based colorimetric assay in assessing photoradiation therapy in vitro. Cancer Lett. 41, 315-321.

19. Shulok, J. R., M. H. Wade and C.-W. Lin (1990) Subcellular localization of hematoporphyrin derivative in bladder tumor cells in culture. Photochem. Photobiol. 51, 45 1 4 5 7 .

20. Prendergast, F. G., R. P. Haugland and P. J. Callahan (1981) 1 - [4-(Trimethylamino)phenyl]-6-phenylhexa- 1,3,5-triene: synthesis, fluorescence properties and use as a fluorescence probe of lipid bilayers. Biochemistry 20, 7333-7338.

21. Kessel, D. (1993) Use of fluorescent probes for characterizing sites of photodamage. The Spectrum 6, 1-6.

22. Kastan, M. B., C. E. Canman and C. J. Leonard (1995) P53, cell cycle control and apoptosis implications. Cancer Metastasis Rev. 14, 3-15.

23. Moan, J. and K. Berg (1992) Photochemotherapy of cancer: experimental research. Photochem. P hotobiol. 55, 93 1-948.

24. Henderson, B. W. and T. J. Dougherty (1992) How does photodynamic therapy work? Photochem. Photobiol. 55, 145-1 57.

25. Hickman, J. A,, C. S . Potten, A. J. Merritt and T. C. Fisher (1994) Apoptosis and cancer chemotherapy. Phil. Trans. R. Soc. Lond. B 345, 3 19-325.

26. Dive, C. and J. A. Hickman (1991) Drug-target interactions: only the first step in the commitment to a programmed cell death? Br. J . Cancer 64, 192-196.

27. Columbano, A. (1995) Cell death: current difficulties in discrim- inating apoptosis from necrosis in the context of pathologic processes in vivo. J . Cell. Biochem. 58, 181-190.

28. Foster, T. H., R. S. Murant, R. G. Bryant, R. S. Knox, S. L. Gibson and R. Hilf (1991) Oxygen consumption and diffusion effects on photodynamic therapy. Radiat. Rex 126, 296-303.

29. Thomas, J. P., R. D. Hall and A. W. Girotti (1987) Singlet

534 Yu Luo eta/.

oxygen intermediacy in the photodynamic action of membrane- bound hematoporphyrin derivative. Cancer Lett. 35, 295-302.

30. He, J., L. R. Friedman, H. E. Larkin, L. Y. Xue, M. L. Agarwal and N. L. Oleinick (1994) Role of bcl-2 expression in conferring resistance to photodynamic therapy (PDT)-induced apoptosis. Photochem. Photobiol. 59S, 48-49.

31. Hockenbery, D. M., Z . N. Oltvai, X.-M. Yin, C. L. Milliman and S. J. Korsmeyer (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241-251.

32. Muschel, R. J. , E. J . Bernhard, L. Garza, W. G. McKenna and C. J. Koch (1995) Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Res. 55, 995-998.

33. Borner, M. M., E. Schneider, F. Pirnia, 0. Sartor, J. B. Trepel and C. E. Myers (1994) The detergent Triton X-100 induces a death pattern in human carcinoma cell lines that resembles cytotoxic lymphocyte-induced apoptosis. FEBS Lett. 353, 129- 132.

rapid initiation of apoptosis by photodynamic therapy

Documents