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.     

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.     

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.     

Pathways to Paraptosis After ER Photodamage in OVCAR‐5 Cells

A death mode termed paraptosis is initiated by photodamage to the endoplasmic reticulum (ER). This is characterized by an extensive pattern of cytoplasmic vacuole formation and leads to a gradual loss of cytoplasm and of viability. Many photosensitizers in clinical use target sub‐cellular organelles that include the ER and can, therefore, invoke paraptosis as well as apoptosis. In this study, we explore pathways to paraptosis in OVCAR‐5, an ovarian cancer cell line of human origin. At low PDT doses, the route to paraptosis follows the pattern that occurs after chemotherapy, that is, a pattern of vacuole formation that can be suppressed by MAPK antagonists or inhibition of new protein synthesis. At PDT doses clinically pertinent for tumor eradication, the pathway to paraptosis appears to be independent of these factors. In addition to morphologic changes, translocation of the nuclear protein HMGB1(high mobility group protein B1) to the cell periphery has been described as a potential marker for drug‐induced paraptosis. This occurs to only a very minor extent after a lethal PDT dose that targets the ER. It appears that a lethal level of ER photodamage can initiate a different pathway to paraptosis, perhaps associated with ER protein cross‐linking.     

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.     

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.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.     

Determinants of the apoptotic response to lysosomal photodamage

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.     

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.     

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.     

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     

Promotion of PDT efficacy by a Bcl-2 antagonist

Photodynamic therapy (PDT) directed against the endoplasmic reticulum (ER) is also known to target antiapoptotic Bcl-2 family proteins. This effect is associated with the initiation of both apoptosis, a cell death pathway, and autophagy, an organelle recycling system that can lead to survival or cell death. In this study, we examined the ability of the Bcl-2 antagonist HA14-1 to promote the photodynamic efficacy of PDT directed at the ER. At concentrations that independently caused only a small loss of viability, HA14-1 markedly enhanced the proapoptotic and phototoxic effects of ER photodamage. These results provide additional evidence that the antiapoptotic properties of Bcl-2 constitute an important determinant of photokilling, and demonstrate that synergistic effects can result when PDT is coupled with pharmacologic suppression of Bcl-2 function.     

GRP78 Induction by Calcium lonophore Potentiates Photodynamic Therapy Using the Mitochondrial Targeting Dye Victoria Blue BO

The cationic photosensitizing triaryl methane dye Victoria Blue BO (VBBO) localizes in mitochondria and causes oxidative damage to this organelle during photodynamic therapy (PDT). Oxidative stresses from other photosensitizers induce a variety of stress proteins. The endoplasmic reticulum (ER)-based, calcium-binding stress protein GRP78 is a putative protective factor for photo-sensitizers such as Photofrin® that damage multiple intracellular sites and for several cytotoxic agents. In the current study VBBO-PDT was found to induce glucose-regulated protein (GRP)78. However, in contrast to other drugs, rather than being protected, human squamous carcinoma cells (FaDu) induced to express GRP78 by calcium ionophore A23187 became more sensitive to PDT. A line of Chinese hamster ovary cells (C.-1) constitutively overexpressing GRP78 also were more sensitive. Cytotoxicity of the A23187 treatment and VBBO was synergistic, with more than 11-fold potentiation with light irradiation, but was only additive in the dark. The in-creased cell killing was not due to differences in VBBO uptake or to changes in the intracellular localization of VBBO caused by calcium ionophore or GRP78. Thus, GRP78 appears to enhance rather than protect against VBBO-induced mitochondrial photodamage and contributes to cell death. This novel finding possibly may stem from the effects of GRP78, ER Ca²⁺ stores and ATP consumption on the Ca²⁺ and ATP-dependent mitochondrial permeability transition that may be evoked by PDT damage to the mitochondrial respiratory chain. The work suggests interventions that may potentiate PDT with mitochondrial targeting sensitizers and potential enhancements in efficacy when GRP78 is upregulated biologically or pharmacologically.     

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.     

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.     

Apoptosis,Paraptosis and Autophagy: Death and Survival Pathways Associated with Photodynamic Therapy

The ability of photosensitizing agents to create photodamage at specific subcellular sites has proved useful for characterizing pathway(s) to cell death and for selecting optimal targets for anti‐tumor efficacy. Both apoptosis and autophagy can occur after photodamage directed at mitochondria, lysosomes or the ER, with the balance often a determinant of overall efficacy. A combination of lysosomal + mitochondrial targets is associated with enhanced efficacy. More recently, ER photodamage was found to evoke a mainly unexplored mode of photokilling that involves extensive cytoplasmic vacuole formation but does not represent autophagy. This has been termed “paraptosis” and appears to be a reaction to the appearance of misfolded ER proteins. This report is designed to summarize current knowledge relating to death pathways and update information relating to paraptosis as a PDT response.     

Highly Efficient,Conjugated‐Polymer‐Based Nano‐Photosensitizers for Selectively Targeted Two‐Photon Photodynamic Therapy and Imaging of Cancer Cells

Two‐photon photodynamic therapy (2P‐PDT) is a promising noninvasive treatment of cancers and other diseases with three‐dimensional selectivity and deep penetration. However, clinical applications of 2P‐PDT are limited by small two‐photon absorption (TPA) cross sections of traditional photosensitizers. The development of folate receptor targeted nano‐photosensitizers based on conjugated polymers is described. In these nano‐photosensitizers, poly{9,9‐bis[6′′‐(bromohexyl)fluorene‐2,7‐ylenevinylene]‐co‐alt‐1,4‐(2,5‐dicyanophenylene)}, which is a conjugated polymer with a large TPA cross section, acts as a two‐photon light‐harvesting material to significantly enhance the two‐photon properties of the doped photosensitizer tetraphenylporphyrin (TPP) through energy transfer. These nanoparticles displayed up to 1020‐fold enhancement in two‐photon excitation emission and about 870‐fold enhancement in the two‐photon‐induced singlet oxygen generation capability of TPP. Surface‐functionalized folic acid groups make these nanoparticles highly selective in targeting and killing KB cancer cells over NIH/3T3 normal cells. The 2P‐PDT activity of these nanoparticles was significantly improved, potentially up to about 1000 times, as implied by the enhancement factors of two‐photon excitation emission and singlet oxygen generation. These nanoparticles could act as novel two‐photon nano‐photosensitizers with combined advantages of low dark cytotoxicity, targeted 2P‐PDT with high selectivity, and simultaneous two‐photon fluorescence imaging capability; these are all required for ideal two‐photon photosensitizers.     

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.     

Albumin–Folate Conjugates for Drug‐targeting in Photodynamic Therapy

Photodynamic therapy (PDT) is based on the cytotoxicity of photosensitizers in the presence of light. Increased selectivity and effectivity of the treatment is expected if a specific uptake of the photosensitizers into the target cells, often tumor cells, can be achieved. An attractive transporter for that purpose is the folic acid receptor α (FRα), which is overexpressed on the surface of many tumor cells and mediates an endocytotic uptake. Here, we describe the synthesis and photobiological characterization of polar β‐carboline derivatives as photosensitizers covalently linked to folate‐tagged albumin as the carrier system. The particles were taken up by KB (human carcinoma) cells within <90 min and then co‐localized with a lysosomal marker. FRα antibodies prevented the uptake and also the corresponding conjugate without folate was not taken up. Accordingly, a folate‐albumin‐β‐carbolinium conjugate proved to be phototoxic, while the corresponding albumin–β‐carbolinium conjugates without FA were nontoxic, both with and without irradiation. An excess of free folate as competitor for the FRα‐mediated uptake completely inhibited the photocytotoxicity. Interestingly, the albumin conjugates are devoid of photodynamic activity under cell‐free conditions, as shown for DNA as a target. Thus, phototoxicity requires cellular uptake and lysosomal degradation of the conjugates. In conclusion, albumin–folate conjugates appear to be promising vehicles for a tumor cell targeted PDT.