Promotion of Proapoptotic Signals by Lysosomal Photodamage

We previously reported that low‐level lysosomal photodamage enhanced the efficacy of subsequent mitochondrial photodamage, resulting in a substantial promotion of apoptotic cell death. We now extend our analysis of the sequential PDT protocol to include two additional lysosomal‐targeting photosensitizers. These agents, because of enhanced permeability, are more potent than the agent (N‐aspartyl chlorin E6, NPe6) used in the initial study. Addition of the cell‐permeable cysteine protease inhibitor E‐64d and calcium chelator BAPTA‐AM almost completely suppressed sequential PDT‐induced loss of mitochondrial membrane potential and activation of procaspases‐3 and ‐7. These inhibitors did not, however, suppress the proapoptotic effect of a BH3 mimetic or mitochondrial photodamage. Knockdowns of ATG7 or ATG5, proteins normally associated with autophagy, suppressed photodamage induced by the sequential PDT protocol. These effects appear to be independent of the autophagic process as pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdowns may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial proapoptotic processes. Our results suggest that low‐dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers.     

Enhanced responsiveness to photodynamic therapy-induced apoptosis after mitochondrial DNA depletion

Several lines of evidence indicate that mitochondria are an especially sensitive target for photodamage. Reports of cross resistance between photodynamic therapy (PDT) and the drug cisplatin, along with evidence that depletion of mitochondrial DNA (mtDNA) sensitized cells to cisplatin suggested a study of the photodynamic responsiveness of murine leukemia control L1210 cells versus cells depleted of mtDNA. Loss of mtDNA led to an increased sensitivity to mitochondrial photodamage, while the cytotoxic effects of lysosomal photodamage were not affected. Cells depleted of mtDNA showed an enhanced apoptotic response to PDT involving a mitochondrial target, compared with control cells.     

Reversible Effects of Photodamage Directed Toward Mitochondria

When the initial effect of photodynamic therapy (PDT) involves mitochondrial photodamage, an early effect is loss of the mitochondrial membrane potential (ΔΨ_m). Using murine hepatoma 1c1c7 cells and a photosensitizing agent known to target mitochondria, we examined loss of ΔΨ_m, initiation of apoptosis and loss of viability as a function of time and light dose. There was a correlation between loss of viability and the rapid disappearance of ΔΨ_m, as detected by the potential‐sensitive probe Mitotracker Orange (MTO). Loss of ΔΨ_m was, however, reversible even with a substantial loss of viability. Unless there was a supralethal level of photodamage, 1c1c7 cells recovered their mitochondrial membrane potential, even if the cell population was on the pathway to apoptosis and cell death. These results indicate that when mitochondria are the initial PDT target, a qualitative estimate of photokilling can be provided by assessing the initial loss of ΔΨ_m.     

Enhanced Efficacy of Photodynamic Therapy via a Sequential Targeting Protocol

This study was designed to examine determinants of the discovery that low‐dose lysosomal photodamage (lyso‐PDT) could potentiate the efficacy of subsequent low‐dose mitochondrial photodamage (mito‐PDT). The chlorin NPe6 and the benzoporphyrin derivative (BPD) were used to separately target lysosomes and mitochondria, respectively, in murine hepatoma cells. Lyso‐PDT (LD₅ conditions) followed by mito‐PDT (LD₁₅ conditions) enhanced the loss of the mitochondrial membrane potential, activation of procaspases‐3/7 and photokilling. Reversing the sequence was less effective. The optimal sequence did not enhance reactive oxygen species formation above that obtained with low‐dose mito‐PDT. In contrast, alkalinization of lysosomes with bafilomycin also enhanced low‐dose mito‐PDT photokilling, but via a different pathway. This involves redistribution of iron from lysosomes to mitochondria leading to enhanced hydroxyl radical formation, effects not observed after the sequential procedure. Moreover, Ru360, an inhibitor of mitochondrial calcium and iron uptake, partially suppressed the ability of bafilomycin to enhance mito‐PDT photokilling without affecting the enhanced efficacy of the sequential protocol. We conclude that sequential PDT protocol promotes PDT efficacy by a process not involving iron translocation, but via promotion of the pro‐apoptotic signal that derives from mitochondrial photodamage.     

Promotion of Proapoptotic Signals by Lysosomal Photodamage: Mechanistic Aspects and Influence of Autophagy

Prior studies demonstrated that a low level (LD_10–15) of lysosomal photodamage can sensitize cells to the apoptotic death that results from subsequent mitochondrial photodamage. We have proposed that this process occurs via a calpain‐catalyzed cleavage of the autophagy‐associated protein ATG5 to form a proapoptotic fragment. In this report, we provide evidence for the postulated ATG5 cleavage and show that the sequential photodynamic therapy (PDT) protocol can also partly overcome the adverse effect of hypoxia on the initiation of apoptosis. While autophagy can offer cytoprotection after mitochondrial photodamage, this does not appear to apply when lysosomes are the target. This may account for the ability of very low PDT doses directed at lysosomes to evoke ATG5 cleavage. The resulting proapoptotic effect overcomes intrinsic cytoprotection from mitochondrial photodamage along with a further stimulation of phototoxicity.     

Effects of endosomal photodamage on membrane recycling and endocytosis

The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically substituted porphine or a phthalocyanine also prevents wortmannin-induced endosomal swelling, even when the photodynamic therapy (PDT) dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers.     

Subcellular Targeting as a Determinant of the Efficacy of Photodynamic Therapy

In prior studies, we have identified the ability of low‐level lysosomal photodamage to potentiate the phototoxic effect of subsequent photodamage to mitochondria. The mechanism involves calpain‐mediated cleavage of the autophagy‐associated protein ATG5 to form a proapoptotic fragment (tATG5). In this report, we explore the permissible time lag between the two targeting procedures along with the effect of simultaneously targeting both lysosomes and mitochondria. This was found to be as effective as the sequential protocol with no gap between the irradiation steps. Inhibition of calpain reversed the enhanced efficacy of the “simultaneous” protocol. It appears that even a minor level of lysosomal photodamage can have a significant effect on the efficacy of subsequent mitochondrial photodamage. We propose that these results may explain the efficacy of Photofrin, a photosensitizing product that also targets both lysosomes and mitochondria for photodamage.     

Evidence that bcl-2 is the target of three photosensitizers that induce a rapid apoptotic response

We originally proposed that the subcellular target for one class of photosensitizing agents was the mitochondrion. This classification was based on effects that occur within minutes of irradiation of photosensitized cells: rapid loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol and activation of caspase-3. These effects were followed by the appearance of an apoptotic morphology within 30-90 min. Fluorescence localization studies on three sensitizers initially classified as 'mitochondrial' revealed that these agents bind to a variety of intracellular membranes. The earliest detectable effect of photodamage is the selective loss of the antiapoptotic protein bcl-2 leaving the proapoptotic protein bax undamaged. Bcl-2 photodamage can be detected directly after irradiation of cells at 10 degrees C. Subsequent warming of cultures to 37 degrees C results in loss of delta psi m, release of cytochrome c and activation of caspase-3. The latter appears to amplify the other two effects. Based on results reported here we propose that the apoptotic response to these photosensitizers is derived from selective photodamage to the antiapoptotic protein bcl-2 while leaving the proapoptotic protein bax unaffected.     

Primary photodamage sites and mitochondrial events after Foscan photosensitization of MCF-7 human breast cancer cells

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.     

PHOTOSENSITIZATION WITH ETIOBENZOCHLORINS AND OCTAETHYLBENZOCHLORINS

The photophysical and photobiological properties of a series of etiobenzochlorins were evaluated in cell culture using murine leukemia L1210 cells. In the series of agents tested, the chlorin-(mono)sulfonate was the most efficacious, the tin chlorin somewhat less so and the tin chlorin-sulfonate much less active. The parent chlorin was essentially inactive at the limit of solubility. Photodamage was assessed by measuring alterations in surface hydro-phobicity ( via a two-phase partitioning procedure), amino acid transport and membrane potential. Additional information was provided from fluorescence microscopy, which was used to identify sites of sensitizer binding and effects of photodamage on the binding patterns of fluorescent probes specific for mitochondria, lysosomes and plasma membranes. Effects of photodamage on fluorescence lifetime distribution of the membrane probe trimethylamino-diphenyl hexatriene were examined. The data obtained were consistent with localization of the parent etiobenzochlorin and tin derivative at lysosomal loci. the chlorin-sulfonate at plasma and mitochondrial membranes and tin-sulfonate at the cell surface.     

Enhancement of lysosomal osmotic sensitivity induced by the photooxidation of membrane thiol groups

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) and examining the lysosomal beta-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75000 to 125000), which was visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.     

Initiation of Apoptosis versus Necrosis by Photodynamic Therapy with Chloroaluminum Phthalocyanine

Abstract— While chloroaluminum phthalocyanine is a highly effective photosensitizer of murine leukemia P388 or L1210 cells, the mode of cell death varies as a function of the PDT dose. When cells were incubated with 0.3 mUM of the sensitizer, a light dose of 45 mJ cm_-2 (670 5 nm) yielded a 90% apoptotic cell population within 60 min. The sensitizer localized throughout the cytoplasm and catalyzed both lysosomal and mitochondrial photodamage at this light dose. Higher light doses yielded progressively more membrane photodamage and inhibited the apoptotic response as determined by the examination of Hochst dye HO 33342-IabeIed nuclei, DNA fragmentation on gels and a poly(adenosylribose) polymerase (PARP)-cleavage assay. Pulse-field gel electrophoresis revealed nonspecific DNA degradation to particles 50 kbp at the higher PDT doses but neither PARP cleavage nor apoptotic nuclei     

A Combination of Visudyne and a Lipid‐anchored Liposomal Formulation of Benzoporphyrin Derivative Enhances Photodynamic Therapy Efficacy in a 3D Model for Ovarian Cancer

A major objective in developing new treatment approaches for lethal tumors is to reduce toxicity to normal tissues while maintaining therapeutic efficacy. Photodynamic therapy (PDT) provides a mechanistically distinct approach to treat tumors without the systemic toxicity of chemotherapy drugs. PDT involves the light‐based activation of a small molecule, a photosensitizer (PS), to generate reactive molecular species (RMS) that are toxic to target tissue. Depending on the PS localization, various cellular and subcellular components can be targeted, causing selective photodamage. It has been shown that targeted lysosomal photodamage followed by, or simultaneous with, mitochondrial photodamage using two different PS results in a considerable enhancement in PDT efficacy. Here, two liposomal formulations of benzoporphyrin derivative (BPD): (1) Visudyne (clinically approved) and (2) an in‐house formulation entrapping a lipid conjugate of BPD are used in combination with direct PS localization to mitochondria, endoplasmic reticulum and lysosomes, enabling simultaneous photodamage to all three organelles using a single wavelength of light. Building on findings by our group, and others, this study demonstrates, for the first time in a 3D model for ovarian cancer, that BPD‐mediated photodestruction of lysosomes and mitochondria/ER significantly enhances PDT efficacy at lower light doses than treatment with either PS formulation alone.     

Ultrastructural damage in photosensitized endothelial cells: dependence on hematoporphyrin delivery pathways.

The subcellular photodamage to endothelial cells in culture, revealed by transmission electron microscopy, was correlated with discrete delivery pathways of hematoporphyrin (HP). Cell detachment from the extracellular matrix, prominent water influx starting at the outer membrane and formation of blebs followed by cell death were the result of photodynamic damage induced by aqueous HP. Serum-bound HP was internalized by endocytosis and accumulated in lysosomal compartments as located after photosensitization. Obstructed lysosomal membranes, degradation of chromatin and swelling of endoplasmic reticulum were revealed in these cells. Red blood cells (RBCs), preincubated with HP, delivered low amounts of the drug to endothelial cells. The photodamage was limited to the nucleus and nucleolus. The role of photosensitizer delivery pathways in cancer cell damage is discussed.     

Roles of porphyrin and iron metabolisms in the δ-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin and photodamage of tumor cells

δ-Aminolevulinic acid (ALA)-induced porphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy. To clarify the mechanisms of the tumor-preferential accumulation of protoporphyrin, we examined the effect of the expression of heme-biosynthetic and -degradative enzymes on the ALA-induced accumulation of protoporphyrin as well as photodamage. The transient expression of heme-biosynthetic enzymes in HeLa cells caused variations of the ALA-induced accumulation of protoporphyrin. When ALA-treated cells were exposed to white light, the extent of photodamage of the cells was dependent on the accumulation of protoporphyrin. The decrease of the accumulation of protoporphyrin was observed in the cells treated with inducers of heme oxygenase (HO)-1. The ALA-dependent accumulation of protoporphyrin was decreased in HeLa cells by transfection with HO-1 and HO-2 cDNA. Conversely, knockdown of HO-1/-2 with siRNAs enhanced the ALA-induced protoporphyrin accumulation and photodamage. The ALA effect was decreased with HeLa cells expressing mitoferrin-2, a mitochondrial iron transporter, whereas it was enhanced by the mitoferrin-2 siRNA transfection. These results indicated that not only the production of porphyrin intermediates but also the reuse of iron from heme and mitochondrial iron utilization control the ALA-induced accumulation of protoporphyrin in cancerous cells.     

Effects of photoinduced membrane rigidification on the lysosomal permeability to potassium ions

Mechanism for the photoinduced increase in the lysosomal K+ permeability is still unknown. In this study, we investigated the effect of photodamage-induced membrane rigidification on the lysosomal K+ permeability by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and by monitoring proton leakage with p-nitrophenol. Membrane fluidity was measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Methylene blue-mediated photodamage to lysosomes decreased their membrane fluidity and increased their K+ permeability. The photoinduced increase in the K+ permeability can be reversed by fluidizing the rigidified lysosomal membranes with benzyl alcohol. The results suggest that the membrane rigidification induced by photodamage may increase lysosomal K+ permeability. This conclusion is supported by the observation that rigidifying lysosomal membranes by the treatment with membrane rigidifier cholesteryl hemisuccinate also enhanced the lysosomal K+ permeability.     

Lysosomes: a possible target for psoralen photodamage

Treatment in vitro of Ehrlich ascites tumor cells or human fibroblasts with 8-methoxypsoralen (8-MOP, 2.4 microM) and UVA irradiation results in a 30% and 60% respectively reduction in lysosomal beta-galactosidase activity in situ. Under identical conditions one 8-MOP adduct was formed per 2 X 10(4) bases of DNA, one 8-MOP adduct was formed per approximately 10(4) tRNA molecules and one per approximately 100 ribosomes. It is suggested that the decrease in lysosomal beta-galactosidase activity is a result of leakage through the lysosomal membrane caused by psoralen-UVA damage of the lipids in the membrane, since no effect was found on beta-galactosidase in vitro. These results indicate that the lysosomes may also be a target for cellular photodamage by 8-methoxy-psoralen.     

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.     

Biological Effects of a Simplified Synthetic Analogue of Ion-Channel-Forming Polytheonamide B on Plasma Membrane and Lysosomes

Polytheonamide B ( 1 ) is a linear 48-mer natural peptide with alternating d - and l -amino acid residues. Compound 1 forms conducting channels for monovalent ions and exhibits potent cytotoxicity against MCF-7 cells. Previously, we reported that nanomolar concentrations of 1 induce plasma membrane depolarization and lysosomal pH disruption, which triggers apoptosis. Here, we report the cellular localization and biological action of a simplified synthetic analogue of 1 , polytheonamide mimic 3 . Compared with 1 , the toxicity of 3 against MCF-7 cells is 16 times weaker. Although its plasma membrane depolarization effect is only 3.6 times lower, more 3 (20-fold) is required to neutralize lysosomal pH. Thus, the effective concentrations for lysosomal neutralization and cytotoxicity by 3 are comparable. These results strongly suggest that the activity of 3 against the lysosomal membrane is more important for apoptotic cell death than its effects on the plasma membrane, and provide valuable information regarding the unique behavior of polytheonamide-based molecules.     

Localization and photodynamic efficacy of two cationic porphyrins varying in charge distributions

Two meso-tetraphenylporphyrin derivatives bearing adjacent: 5,10-di[4-(N-trimethylaminophenyl)-15,20-diphenylporphyrin (DADP-a) or opposite: 5,15-di[4-(N-trimethylaminophenyl)-10,20-diphenylporphyrin (DADP-o) cationic-N-(CH3)3+ groups on two of the para-phenyl positions were examined with regard to photodynamic properties as a function of charge distribution. The two adjacent positive charges in the DADP-a structure result in a molecular distortion (asymmetry), likely from electrostatic repulsion. This could be responsible for the unusual interaction of this compound with some solvents and detergent micelles. In contrast, DADP-o is a much more symmetric molecule. In a cellular environment, fluorescence spectra of the two agents were essentially identical. Subcellular localization played a major role in photodynamic efficacy. DADP-a localized in mitochondria, and irradiation of photosensitized cells (640-650 nm) resulted in a rapid loss of the mitochondrial membrane potential (delta(psi)m), usually a prelude to apoptotic cell death. In contrast, DADP-o localized in lysosomes, and extensive lysosomal photodamage was observed after irradiation. Both steady-state accumulation levels and absorbance spectra favored DADP-o, but the light dose required for a 90% cell kill was two-fold greater for DADP-o than for DADP-a, at a constant extracellular sensitizer concentration. These data indicate that, on a photons/cell basis, DADP-a was five-fold more efficacious. Fluorescence emission spectra in different solvents and detergents demonstrated a tendency for DADP-a association. We interpret these results to indicate partition of both drugs to membrane loci, with mitochondriabeing the more lethal site for photodamage.