Photodynamic Therapy-induced Apoptosis in Epidermoid Carcinoma Cells REACTIVE OXYGEN AND MITOCHONDRIAL INNER MEMBRANE PERMEABILIZATION*

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. How-ever, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670–675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT

In photodynamic therapy (PDT) 1 , visible light activates a photosensitizing drug accumulated in tumor or other abnormal tissue (1)(2)(3). The interaction between the excited photosensitizer and molecular oxygen produces singlet oxygen ( 1 O 2 ) as well as other reactive oxygen species (ROS) to induce cell death (4 -8). Most common photosensitizers are porphyrins and porphyrin-related macrocycles that are lipophilic and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria (9,10).
The mode of cell death by PDT is often apoptosis, and substantial evidence supports the involvement of mitochondria in this process (10 -12). During apoptosis, mitochondria release at least two soluble proteins from the intermembrane space into the cytosol. These proteins are cytochrome c and apoptosisinducing factor, which initiate downstream apoptotic signaling events (13)(14)(15)(16). The mechanisms by which pro-apoptotic proteins are released from the mitochondria remain controversial. Some studies show that loss of mitochondrial membrane potential (⌬⌿), swelling, and mitochondrial outer membrane rupture are responsible for cytochrome c release (17,18). Other evidence, however, suggests that cytochrome c release can occur without mitochondrial depolarization, and proposals have been made that cytochrome c is released through a specific channel in the mitochondrial outer membrane independent of inner membrane depolarization (19,20).
One explanation for mitochondrial depolarization during cell injury is the onset of the mitochondrial permeability transition (MPT). The MPT is a consequence of the opening of permeability transition (PT) pores in the mitochondrial inner membrane through which solutes less than 1,500 daltons pass (21)(22)(23). As a consequence of PT pore opening, mitochondria uncouple and undergo large amplitude swelling. The outer membrane ruptures during swelling to cause the release of mitochondrial intermembrane proteins, such as cytochrome c and apoptosisinducing factor (24). A number of factors regulate the PT pore. For instance, increased mitochondrial matrix Ca 2ϩ , P i , and ROS induce the PT pore opening, whereas cyclosporin A (CsA), an immunosuppressive cyclic endecapeptide, and trifluoperazine block pore conductance (25). The effect of CsA may be transitory, and more long lasting PT pore blockade is achieved when CsA is combined with trifluoperazine (26,27).
The phthalocyanine photosensitizer Pc 4 preferentially binds to the endoplasmic reticulum, Golgi complex, and mitochondria in L5178Y-R (LY-R) mouse lymphoma cells (28) and induces rapid apoptotic cell death after exposure to light (29). The critical early events and specific subcellular targets triggering PDT-induced apoptotic death remain to be determined. Therefore, the aim of this study was to investigate the role of mitochondria in PDT-induced apoptotic death and specifically to determine whether PT pores are involved in this process. In human epidermoid carcinoma A431 cells, our data indicate that PDT with Pc 4 promotes mitochondrial ROS generation, which leads to mitochondrial depolarization, inner membrane permeabilization, swelling, release of cytochrome c, PARP cleavage, and apoptotic cell death. These events were blocked by a combination of CsA and trifluoperazine, two inhibitors of the MPT, and desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation. These data support the conclusion that PDT-induced mitochondrial ROS initiate onset of a CsA/ trifluoperazine-sensitive mitochondrial inner membrane permeabilization and activate the mitochondrial pathway of apoptotic death.
Photodynamic Therapy (PDT)-The phthalocyanine photosensitizer Pc 4 was kindly provided by Drs. Ying-Syi Li and Malcolm E. Kenney (Department of Chemistry, Case Western Reserve University). Stock solutions (0.5 mM) of Pc 4 were prepared in NЈ,NЈ-dimethylformamide and stored at 4°C. Unless otherwise indicated, cells were incubated with 250 nM Pc 4 in complete culture medium for 3 h at 37°C in the dark and subsequently irradiated with red light using a light-emitting diode array (EFOS, Mississauga, Ontario, Canada) at a fluence of 150 mJ/cm 2 (1 milliwatt/cm 2 , max ϳ670 -675 nm) at room temperature.
Determination of Clonogenicity-Cells were exposed to PDT and immediately harvested by trypsinization. Aliquots of cell suspensions were plated onto 60-mm Petri dishes in amounts sufficient to yield 50 -100 colonies per dish. After 7-9 days, colonies were stained with 0.1% crystal violet in 20% ethanol and counted by eye.
Assessment of Cell Viability-To assess the changes in nuclear morphology typical of apoptosis, A431 cells were cultured on 35-mm glassbottomed dishes (MatTek Corp., Ashland, MA). At different time points following PDT, cells were washed twice with phosphate-buffered saline (PBS), pH 7.4. Subsequently, cells were fixed and permeabilized with 1 ml of 100% methanol at Ϫ20°C for 15 min. After rinsing twice with PBS, cells were stained with 1 M Hoechst dye 33342 (Molecular Probes) for 15 min at room temperature. Cells were then washed twice with PBS and subsequently immersed in a mounting medium containing an antifade reagent (Molecular Probes) and visualized under a Leitz Laborluxs fluorescence microscope with a 350-nm band pass excitation filter and a 450 -490 nm band pass emission filter. The dye stains the nuclei of all cells. The percentage of apoptotic cells displaying nuclear condensation or fragmentation was counted in 3 different fields (a total of 200 cells per treatment group).
To assess apoptosis and necrosis in the same cell population, cells were first incubated with 50 M propidium iodide. Propidium iodide stains the nuclei of cells whose plasma membrane is leaky, typically a hallmark of necrosis. After counting propidium iodide-stained nuclei, cells were incubated with 40 M digitonin, which permeabilizes the plasma membrane allowing all nuclei to be stained by propidium iodide. The percentage of apoptotic cells displaying nuclear condensation or fragmentation was then counted.
Confocal Microscopy-For confocal microscopy, cells were cultured on glass-bottomed 35-mm Petri dishes. To investigate the localization of Pc 4, cells were treated with 250 nM Pc 4 in complete culture medium at 37°C for 3 h. To co-localize Pc 4 to the mitochondria, cells were coloaded with 100 nM MitoTracker Green CMXRos (Molecular Probes) for 15 min at room temperature in complete culture medium. A 63ϫ N.A. 1.4 oil immersion planapochromat objective with a Zeiss 410 confocal microscope was used for all experiments. Confocal images of Pc 4 fluorescence were collected using a 568-nm excitation light from an argon/krypton laser, a 560-nm dichroic mirror, and a 590-nm long pass filter. Images of green MitoTracker fluorescence were collected using a 488-nm excitation light from the argon/krypton laser, a 560-nm dichroic mirror, and a 500 -550 nm band pass barrier filter.
To assess mitochondrial inner membrane permeabilization, Pc 4-treated cells were loaded with 1 M calcein acetoxymethyl (AM) ester (Molecular Probes) for 30 min at room temperature in KRH buffer supplemented with 1 mM CoCl 2 (33). After loading, cells were washed twice with fresh KRH buffer omitting calcein and CoCl 2 . Images of green calcein fluorescence were collected using the same filter settings as for MitoTracker Green CMXRos.
To monitor mitochondrial swelling, Pc 4-treated cells were loaded with 100 nM MitoTracker Red CMXRos for 30 min at 37°C in KRH buffer. After loading, cells were washed twice with fresh KRH. Images of the MitoTracker Red fluorescence were collected over time.
To monitor mitochondrial ⌬⌿, Pc 4-treated cells were loaded with 500 nM rhodamine 123 (Molecular Probes) for 30 min at 37°C in KRH buffer (34). After loading, cells were washed twice with fresh KRH and resuspended in KRH buffer containing 25 nM rhodamine 123. Images of rhodamine 123 were collected using the same filter settings as for MitoTracker Green CMXRos.
To assess cytochrome c release, cells were washed twice with PBS, pH 7.4, after PDT exposure and subsequently fixed and permeabilized with 1 ml of 1% paraformaldehyde at room temperature for 15 min. After rinsing with PBS, cells were incubated in IFB (PBS containing 1% bovine serum albumin and 0.1% Tween 20) buffer for 10 min before incubation in IFB containing mouse anti-cytochrome c antibody (1:100 dilution, clone 6H2.B4, PharMingen) for 1 h at room temperature or overnight at 4°C. After rinsing with IFB to remove unbound antibody, cells were incubated in IFB containing horse anti-mouse IgG conjugated to Texas Red (1:60 dilution, Vector Laboratories) for 1 h at room temperature or overnight at 4°C. Cells were then stained with 1 M Hoechst dye 33342 (Molecular Probes) before confocal images were collected using the same filter settings as for MitoTracker Red CMXRos. Fluorescence of Hoechst dye was imaged using 364-nm excitation light from a UV/argon laser, a 460-nm dichroic mirror, and a 417-482 nm band pass barrier filter.

Pc 4 Is Localized to
Mitochondria-We showed previously that Pc 4 preferentially, but not exclusively, binds to the mitochondria of LY-R mouse lymphoma cells (28). To determine whether the binding pattern of Pc 4 is cell type-specific, we loaded human epidermoid carcinoma A431 cells with Pc 4 in complete medium. Because the absorption spectrum of Pc 4 is considerably broadened upon binding to cellular components, Pc 4 within cells can be excited at 568 nm with a em of Ն590 nm. Fluorescence of Pc 4 was imaged using laser scanning confocal microscopy. As shown in Fig. 1 (left panel), Pc 4 displayed a punctate pattern of fluorescence primarily localized to the perinuclear area. To assess whether Pc 4 binds to the mitochondria, cells were co-loaded with MitoTracker Green CMXRos, a mitochondria-specific dye. The bright, punctate fluorescence shown in the Pc 4 image (Fig. 1, left panel) corresponded to MitoTracker Green fluorescence ( Fig. 1, right panel), indicating mitochondrial localization of Pc 4. However, Pc 4 fluorescence did not exclusively correspond to MitoTracker Green fluorescence, indicating that Pc 4 also binds to other intracellular organelles, presumably Golgi complexes and endoplasmic reticulum, as previously shown in LY-R cells (28).
Interestingly, Pc 4 was never found localized to the plasma membrane in either LY-R or A431 cells (Fig. 1, left panel).
Photodynamic Therapy Induces Apoptosis in A431 Cells-By having established that Pc 4 localizes preferentially to mitochondrial membranes, we next determined a dose of PDT that would yield approximately 1 log of cell killing. A431 cells were loaded with 250 nM Pc 4 for 3 h and subsequently irradiated with various fluences of light. Light at 150 mJ/cm 2 reduced the survival by ϳ90% (LD 90 ), as assessed by a clonogenic assay ( Fig. 2A). Therefore, this dose of PDT (250 nM Pc 4, 150 mJ/cm 2 of 670 -675 nm light) was chosen for further experiments. To demonstrate that this dose of PDT causes apoptosis, we monitored PARP cleavage and changes in nuclear morphology characteristic of apoptosis. One h after Pc 4-PDT, about 40% of PARP was cleaved, whereas complete cleavage occurred after 2 h (Fig. 2B). Cells began to display nuclear condensation and fragmentation by 4 h, as indicated by Hoechst 33342 staining (Fig. 2C, arrows).
Photodynamic Therapy Induces Mitochondrial ROS Generation-By using the LD 90 dose of Pc 4-PDT as determined in previous experiments, we next monitored intracellular ROS formation, as measured by the conversion of non-fluorescent 2Ј,7Ј-dichlorofluorescin to fluorescent 2Ј,7Ј-dichlorofluorescein (DCF). After Pc 4-PDT, DCF fluorescence increased within 5 min (Fig. 3A, ϩPc 4). Region-of-interest analysis revealed a 300% increase of fluorescence over control (Fig. 3B, p Ͻ 0.001). Red light alone without Pc 4 caused a small increase in fluorescence possibly due to illumination of the sample by the DCF excitation light from the argon laser (Fig. 3A, ϪPc 4, and B, ϪPc 4, 5 min). To confirm that increased DCF fluorescence after Pc 4-PDT originated from mitochondria, we co-loaded cells with a mitochondria-specific dye, MitoTracker Red. Because the fluorescence of MitoTracker Red was much brighter than that of Pc 4, we were able to observe MitoTracker Red fluorescence in the presence of Pc 4 using the same filter settings as for Pc 4 but with a higher attenuation of the illuminating laser light. The punctate perinuclear fluorescence of DCF co-localized with MitoTracker Red fluorescence, indicating that ROS were mostly formed in mitochondria (Fig. 3C).
Desferrioxamine (500 M), an iron chelator that inhibits iron-mediated hydroxyl radical formation from superoxide and hydrogen peroxide, prevented mitochondrial ROS production (Fig. 3B, ϩPc 4 ϩ DFO). Region-of-interest analysis revealed that desferrioxamine suppressed the increase of DCF fluorescence after Pc 4-PDT from 300 to 50%, the same increase as occurred after light exposure without Pc 4 (Fig. 3B, p Ͻ 0.001). Besides desferrioxamine, sodium azide also prevented ROS production (Fig. 3B), suggesting that the mitochondrial ROS generation was a result of 1 O 2 produced by Pc 4-PDT. In contrast, a combination of CsA (1 M) and trifluoperazine (1 M), inhibitors of the MPT, did not prevent mitochondrial ROS production. Similarly, the caspase-3 inhibitor Ac-DEVD-CHO (100 M) did not prevent mitochondrial ROS production (Fig. 3B).
Photodynamic Therapy Induces Mitochondrial Depolarization-We recently showed that Pc 4-PDT causes rapid inhibition of mitochondrial respiration, as measured in digitoninpermeabilized LY-R mouse lymphoma cells (35). We examined whether the initial mitochondrial ROS production after Pc 4-PDT alters mitochondrial ⌬⌿. A431 cells were loaded with rhodamine 123 to monitor mitochondrial ⌬⌿ in single cells by confocal microscopy. Polarized mitochondria were imaged as bright fluorescent spheres and tubes (Fig. 4A, 0 min). After Pc 4-PDT a majority of the bright spheres disappeared, indicating mitochondrial depolarization (Fig. 4A, ϩPDT, 5-30 min). Noteworthy was the observation that mitochondria depolarized one at a time. Thus, 5 min after Pc 4-PDT, some of the mitochondria with their rhodamine 123 fluorescence disappeared completely, but a subpopulation of fully polarized mitochondria remained. Area-of-interest analysis revealed 60% loss of rhodamine 123 fluorescence by 30 min after PDT (Fig. 4B). The residual 40% of cellular rhodamine fluorescence remaining 30 min after PDT likely reflects the combination of unquenching effect of rhodamine 123 due to leakage of the dye from mitochondria to the cytosol and retention of the dye within cells due to the plasma membrane potential. Addition of carbonyl cyanide p-chlorophenylhydrazone (5 M), a mitochondrial uncoupler, decreased rhodamine 123 fluorescence to 5%. Carbonyl cyanide p-chlorophenylhydrazone also collapses plasma membrane potential and therefore allows rhodamine 123 to leak out from the cells resulting in further decrease of rhodamine 123 fluorescence. This remaining 5% fluorescence likely represents unspecific binding of rhodamine 123 (36). In contrast, mitochondria did not depolarize when incubated with Pc 4 in the absence of light (Fig. 4, ϪPDT).
To test the ability of desferrioxamine and CsA plus trifluoperazine to prevent the decline of mitochondrial ⌬⌿, cells were loaded with rhodamine 123 and subjected to Pc 4-PDT in the presence of desferrioxamine or CsA plus trifluoperazine. Desferrioxamine completely blocked mitochondrial depolarization (Fig. 4, A and B). CsA plus trifluoperazine also protected against mitochondrial depolarization, although to a slightly lesser extent than desferrioxamine (Fig. 4, A and B).
Photodynamic Therapy Induces Mitochondrial Inner Membrane Permeabilization-Because CsA plus trifluoperazine delayed mitochondrial depolarization after Pc 4-PDT, we assessed whether depolarization is due to mitochondrial inner membrane permeabilization, the event that occurs during onset of the MPT. Accordingly, A431 cells were loaded with calcein AM (1 M) and CoCl 2 (1 mM) at room temperature for 30 min (33). Calcein AM diffuses across the plasma membrane and mitochondrial membranes and subsequently becomes deesterified and trapped within the cytoplasm and mitochondria. CoCl 2 is transported into the cytosol where it forms a complex with calcein, quenching its fluorescence. Because CoCl 2 does not enter the mitochondria, calcein fluorescence in the mitochondrial matrix is shown as bright fluorescent spots in confocal images, indicating that mitochondria are impermeable to low molecular weight solutes (Fig. 5A). After Pc 4-PDT, calcein began to leak out from mitochondria to the cytosol; by 30 min only 30% of the calcein remained in the mitochondria, demonstrating permeabilization of the inner mitochondrial membrane (Fig. 5, A and B, ϩPDT). The remaining 30% of calcein fluorescence likely reflects leakage of calcein from mitochondria to the cytosolic space resulting in diffuse fluorescence. This fluorescence contributes to fluorescence intensity within any given region-of-interest. The same phenomenon was observed in earlier reports by Scorrano et al. (37). Desferrioxamine almost completely blocked calcein release from mitochondria (Fig. 5, A and B, ϩPDT/DFO). Likewise, the combination of CsA and trifluoperazine prevented calcein release, although less effectively than desferrioxamine. This last finding implicates the involvement of the MPT in PDT-induced mitochondrial depolarization and inner membrane permeabilization.
PDT Induces Mitochondrial Swelling-The MPT causes large amplitude mitochondrial swelling (24). Therefore, we investigated whether PDT-induced inner membrane permeabilization is associated with mitochondrial swelling. MitoTracker Red is taken up by polarized mitochondria. Once inside mitochondria, MitoTracker Red binds covalently to sulfhydryls and is retained by mitochondria after depolarization, unlike rhodamine 123 or tetramethylrhodamine methyl ester which are released. Therefore, MitoTracker Red is a suitable dye to monitor mitochondrial volume changes under conditions that produce mitochondrial depolarization. Before PDT, MitoTracker Red-labeled mitochondria were bright fluorescent spheres and rods (Fig. 6, ϪPDT). By 15 min after Pc 4-PDT, mitochondrial morphology changed; mitochondria began to round up and increase in diameter (Fig. 6, ϩPDT). Mitochondrial swelling eventually caused leakage of MitoTracker Red from the mitochondria to the cytosol. As a result of this, the cytosolic space displayed diffuse fluorescence, as shown in the right panel of Fig. 6. Onset of swelling closely followed mitochondrial inner membrane permeabilization and depolarization (Figs. 4 and 5).
Blockade of Cytochrome c Release by CsA Plus Trifluoperazine and Desferrioxamine-Although mechanisms underlying cytochrome c release from mitochondria to the cytosol remain unresolved, the opening of mitochondrial PT pores has been suggested as one of the mechanisms (24). Because CsA plus trifluoperazine and desferrioxamine prevented calcein release from mitochondria, we investigated whether these reagents also block cytochrome c release, as assessed by immunocytochemistry. Before Pc 4-PDT, cytochrome c was confined to mitochondria, as indicated by a punctate fluorescence (Fig. 7A,  0 min). By 30 min after Pc 4-PDT, cytochrome c began to leak out from individual mitochondria to the cytosol, resulting in diffuse fluorescence (Fig. 7, A and B). After 120 min, 80% of the cells displayed diffuse fluorescence (data not shown). CsA plus trifluoperazine or desferrioxamine blocked cytochrome c release. Sixty min after PDT, 41.0 Ϯ 3.1% (n Ϯ S.E.) of cells displayed diffuse fluorescence compared with 21.3 Ϯ 0.7 and 21.1 Ϯ 1.6% (n Ϯ S.E.) in the presence of CsA plus trifluoperazine and desferrioxamine, respectively (Fig. 7B). In addition, CsA plus trifluoperazine or desferrioxamine blocked most PDTinduced apoptosis assessed by changes in nuclear morphology (Fig. 8). A small percentage of cells also died by necrosis assessed by propidium iodide uptake. However, necrotic death was not decreased by CsA plus trifluoperazine or desferrioxamine.
Caspase-3 Inhibition Does Not Prevent Mitochondrial Depolarization but Blocks Apoptosis-Some reports in the literature suggest that cytochrome c release occurs without mitochondrial depolarization. Rather, caspase-3 activation following cytochrome c release may result in mitochondrial depolarization, because caspase-3 inhibition prevented depolarization but not cytochrome c release (38). To test this possibility, we monitored mitochondrial ⌬⌿ in the presence of Ac-DEVD-CHO, a caspase-3 inhibitor. The results show that Ac-DEVD-CHO (100 M) did not prevent mitochondrial ROS generation (Fig. 3C) or depolarization (Fig. 8A) but did inhibit apoptosis (Fig. 8B). As with CsA plus trifluoperazine or DFO, the most apparent difference with or without Ac-DEVD-CHO is the number of apoptotic cells, whereas the number of necrotic cells remains constant (7-10%). These results demonstrate that caspase-3 activation is a downstream event from mitochondrial depolarization in Pc 4-PDT-induced cell death. DISCUSSION Our results show the temporal sequence of events in mitochondria during apoptosis induced by PDT in single living epidermoid carcinoma cells. PDT with Pc 4 triggers mitochondrial ROS production resulting in inner membrane permeabilization, mitochondrial depolarization, and swelling, which in turn leads to cytochrome c release and apoptotic death. Blockade of mitochondrial ROS production with desferrioxamine prevented all these events, whereas inhibition of PT pores with CsA plus trifluoperazine blocked mitochondrial inner membrane permeabilization and subsequent apoptosis but did not block ROS formation. These results suggest a causal link between mitochondrial ROS, mitochondrial inner membrane permeabilization, and apoptotic death in response to Pc 4-PDT.
Most PDT photosensitizers are porphyrins or porphyrinrelated macrocycles that are hydrophobic and accumulate in cellular membranes (9). The binding of PDT photosensitizers to mitochondria has been associated with efficient induction of apoptosis (10). Pc 4 is a second generation photosensitizer that is a highly efficient inducer of apoptosis in murine lymphoma LY-R cells (5) and human tumor cells (39). Previously, we showed (28) that Pc 4 binds preferentially to mitochondria, endoplasmic reticulum, and Golgi complex but not to the plasma membrane in LY-R cells. In the present study, we confirmed a strong mitochondrial localization of Pc 4 also in epidermoid carcinoma A431 cells (Fig. 1). Another photosensitizer, hematoporphyrin IX, has been shown to accumulate preferentially in protein regions of the mitochondrial inner membrane as assessed by fluorescence anisotropy of isolated mitochondria (40,41). Although the lateral spatial resolution of confocal microscopy limits identification of the exact binding site of Pc 4, the probable binding sites are lipid and protein regions on the outer and inner mitochondrial membranes. Recently, we reported that Bcl-2, a protein of the mitochondrial outer membrane, is specifically photodamaged by Pc 4-PDT (42). This observation is consistent with a Pc 4-binding site on the outer membrane in the immediate vicinity of Bcl-2.
Light activation of photosensitizers produces 1 O 2 and other ROS in cells (4). We showed previously (43) that Pc 4 is capable of generating 1 O 2 . 1 O 2 is very reactive and capable of damaging nucleic acids, proteins, and lipids (44). Because the lifetime of 1 O 2 is very short, damage caused by 1 O 2 occurs in close proximity to the binding site of the photosensitizer (45). In the present study, Pc 4-PDT caused a large increase of ROS generation (3-fold increase) as revealed by confocal microscopy from the conversion of non-fluorescent dichlorofluorescin to the highly fluorescent dichlorofluorescein (Fig. 3). Most ROS generation occurred within mitochondria (Fig. 3C). Dichlorofluorescin does not detect ROS that can be detected with dichlorofluorescin. The previous observation (43) that Pc 4 induces 1 O 2 generation and the observation in the present work that sodium azide, a specific scavenger of 1 O 2 , blocked the increase of DCF fluorescence (Fig. 3B) suggest that 1 O 2 was the initial ROS formed after PDT. The fact that the iron chelator, desferrioxamine, also strongly inhibited mitochondrial ROS formation detected by dichlorofluorescein fluorescence suggests that these ROS were formed by an iron-dependent mechanism within mitochondria, likely the ironcatalyzed Haber-Weiss reaction to form hydroxyl radicals or lipid hydroperoxides. CsA plus trifluoperazine did not prevent mitochondrial ROS formation (Fig. 3B), which indicates that onset of the MPT was not the cause of increased ROS formation.
PDT induced mitochondrial inner membrane permeabilization and concomitant mitochondrial depolarization in A431 cells (Figs. 4 and 5). The confocal images in Fig. 4 suggest that depolarization of individual mitochondria was an all-or-nothing event. Overall rhodamine 123 fluorescence was decreased by 60% at 30 min (Fig. 4B). Some of the remaining fluorescence may represent nonspecific rhodamine 123 binding, and some fluorescence reflects the cytosolic rhodamine 123 retained within cells by the plasma membrane potential. By 30 min after Pc 4-PDT, about 30% of calcein fluorescence remained (Fig. 5B). This remaining fluorescence likely reflects the diffuse cytosolic calcein fluorescence after onset of the MPT (25). Taken together, the rhodamine 123 and calcein data indicate that Pc 4-PDT induces mitochondrial inner membrane permeabilization. The opening of the PT pores is regulated by mitochondrial ⌬⌿. A drop in mitochondrial gating potential promotes the opening of the PT pores (47). After Pc 4-PDT, an initial drop in mitochondrial ⌬⌿ may help promote the inner membrane permeabilization. Then a positive feedback loop may be activated whereby mitochondrial inner membrane permeabilization further promotes mitochondrial depolarization.
Previously, we showed (35) that PDT induces inhibition of mitochondrial respiration and cytochrome c release in digitonin-permeabilized cells. Mitochondrial respiratory inhibition was reversed by addition of exogenous cytochrome c. These data indicate that PDT does not cause direct damage to the respiratory components and further support the results of the present study.
During the initial 15 min after PDT, both mitochondrial depolarization and calcein release occurred over similar time courses (Figs. 4 and 5). We were not able to monitor mitochondrial depolarization and inner membrane permeabilization simultaneously in the same cells due to spectral overlap of the fluorophores. Therefore, it is difficult to assess the exact temporal sequence of events because initial fluorescence changes occurred so rapidly. Based on comparison of separate experiments, inner membrane permeabilization (calcein release) and mitochondrial depolarization (rhodamine 123 release) occurred almost simultaneously.
Recently, photoactivation of two types of porphycenes was reported to induce an immediate loss of mitochondrial ⌬⌿ in murine leukemia cells (12). In this study, mitochondrial ⌬⌿ was monitored with Mitotracker Orange TM , which by itself inhibits the mitochondrial respiratory chain at complex I and induces onset of the MPT (48). In mouse lymphoma LY-R cells, a PDT dose of 300 nM Pc 4 and 120 mJ/cm 2 of red light induced rapid cytochrome c release accompanied by mitochondrial depolarization as measured by JC-1 fluorescence (49). Interestingly, lower doses of PDT (e.g. light doses Յ90 mJ/cm 2 ) were shown to induce cytochrome c release without the loss of ⌬⌿, suggesting that the release of cytochrome c from mitochondria resulting from PDT is independent of the loss of ⌬⌿. Differences in PDT dose and cell type may explain the differences in the results.
In the present study, cytochrome c release and apoptosis followed mitochondrial inner membrane permeabilization and depolarization (Figs. 7 and 8). CsA plus trifluoperazine or desferrioxamine blocked these events, showing a critical role of mitochondrial ROS and subsequent MPT in the induction of apoptotic signaling after Pc 4-PDT. A recent report (38) suggests that mitochondrial depolarization occurs downstream of caspase-3 activation in the apoptotic pathway. This conclusion was based on the observation that caspase-3 inhibition prevented mitochondrial depolarization but did not block cytochrome c release. However, in the present study, caspase-3 inhibition blocked apoptosis but did not prevent mitochondrial ROS production and depolarization. A small percentage of cells also died by necrosis; however, caspase inhibition had no effect on this component of cell killing. These findings indicate that mitochondrial depolarization was upstream of caspase-3 activation in the Pc 4-PDT apoptotic pathway.
Onset of the MPT is implicated in several models of necrotic and apoptotic cell death (32, 50 -55). Although details of the regulation of the PT pore complex are still uncertain, evidence indicates that ROS promote PT pore opening via oxidation of matrix glutathione (56,57). Opening of the PT pores results in mitochondrial inner membrane permeabilization, mitochondrial uncoupling, and swelling. This swelling leads to outer membrane rupture, which causes mitochondrial intermembrane proteins to be released into the cytosol (24). Two pharmacological agents, CsA and trifluoperazine, have been extensively used to inhibit the MPT (18,26,27,58). In the present study, the combination of CsA and trifluoperazine prevented substantially inner membrane permeabilization and mitochondrial depolarization without blocking mitochondrial ROS production (Figs. 4 and 5). This places the step inhibited by CsA/trifluoperazine downstream of desferrioxamine-sensitive ROS formation in the apoptotic cascade.
In conclusion, we propose the following sequence of events in response to PDT with Pc 4 in human epidermoid carcinoma A431 cells (Fig. 9). The initial response to PDT is formation of azidesensitive 1 O 2 in mitochondria. Subsequently, iron-dependent reactions lead to lipid preoccupation. Mitochondrial ROS further induce mitochondrial inner membrane permeabilization resulting in mitochondrial depolarization, swelling, cytochrome c release, and apoptotic death. Blockade of mitochondrial ROS production and inhibition of mitochondrial inner membrane permeabilization protect against PDT-induced apoptotic death. Overall, our data show that mitochondrial ROS formation plays a critical role in PDT-induced apoptosis.