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.     

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.     

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.     

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.     

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.     

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.     

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.     

Metal-Organic Layer Delivers 5-Aminolevulinic Acid and Porphyrin for Dual-Organelle-Targeted Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) depends on the subcellular localization of photosensitizers. Herein, we report a dual-organelle-targeted nanoparticle platform for enhanced PDT of cancer. By grafting 5-aminolevulinic acid (ALA) to a Hf₁₂-based nanoscale metal-organic layer (Hf-MOL) via carboxylate coordination, ALA/Hf-MOL enhanced ALA delivery and protoporphyrin IX (PpIX) synthesis in mitochondria, and trapped the Hf-MOL comprising 5,15-di-p-benzoatoporphyrin (DBP) photosensitizers in lysosomes. Light irradiation at 630 nm simultaneously excited PpIX and DBP to generate singlet oxygen and rapidly damage both mitochondria and lysosomes, leading to synergistic enhancement of the PDT efficacy. The dual-organelle-targeted ALA/Hf-MOL outperformed Hf-MOL in preclinical PDT studies, with a 2.7-fold lower half maximal inhibitory concentration in cytotoxicity assays in vitro and a 3-fold higher cure rate in a colon cancer model in vivo.     

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.     

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.     

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.     

GSH and H2O2 Co‐Activatable Mitochondria‐Targeted Photodynamic Therapy under Normoxia and Hypoxia

Currently, photosensitizers (PSs) that are microenvironment responsive and hypoxia active are scarcely available and urgently desired for antitumor photodynamic therapy (PDT). Presented herein is the design of a redox stimuli activatable metal‐free photosensitizer (aPS), also functioning as a pre‐photosensitizer as it is converted to a PS by the mutual presence of glutathione (GSH) and hydrogen peroxide (H₂O₂) with high specificity on a basis of domino reactions on the benzothiadiazole ring. Superior to traditional PSs, the activated aPS contributed to efficient generation of reactive oxygen species including singlet oxygen and superoxide ion through both type 1 and type 2 pathways, alleviating the aerobic requirement for PDT. Equipped with a triphenylphosphine ligand for mitochondria targeting, ^mito aPS showed excellent phototoxicity to tumor cells with low light fluence under both normoxic and hypoxic conditions, after activation by intracellular GSH and H₂O₂. The ^mito aPS was also compatible to near infrared PDT with two photon excitation (800 nm) for extensive bioapplications.     

Effects of HPV Status on Responsiveness to Ionizing Radiation vs Photodynamic Therapy in Head and Neck Cancer Cell lines

Efficacy of ionizing radiation (I/R) was compared with phototoxic effects of photodynamic therapy (PDT) in vitro using two cell lines derived from patients with head and neck squamous cell carcinoma (HNSCC). A cell line derived from a donor with a human papilloma virus (HPV) infection was more responsive to I/R but significantly less responsive to PDT than a cell line derived from an HPV-free patient. Cell death after I/R in the HPV(+) cell line was associated with increased DEVDase activity, a hallmark of apoptosis. The HPV(−) line was considerably less responsive to I/R, with DEVDase activity greatly reduced, suggesting an impaired apoptotic program. In contrast, the HPV(−) cells were readily killed by PDT when the ER was among the targets for photodamage. While DEVDase activity was enhanced, the death pathway appears to involve paraptosis until the degree of photodamage reached the LD₉₉ range. These data suggest that PDT-induced paraptosis can be a death pathway for cells with an impaired apoptotic program.     

Universal Scaffold for an Activatable Photosensitizer with Completely Inhibited Photosensitivity

Activatable photosensitizers (aPSs) sensitive to specific stimuli hold potential for targeting multiple disease biomarkers and are desirable for photodynamic therapy (PDT). Presented herein is the design of aPSs that can be activated and fully recover from an inhibited state in the presence of specific biomarker. A designed long‐wavelength D‐π‐A photosensitizer, PS_Se‐I , with highly efficient photosensitivity for generation of ¹O₂ was used. Caging of the phenolate donor of PS_Se‐I with a biomarker‐sensitive group provided ^ALP PS , and the drastic activation of its photosensitivity was demonstrated intracellularly. To enhance the flexibility of the design strategy, a clickable azide group was introduced into the scaffold to allow integration of more functionality. Modularly derived ^mito‐PN PS , equipped with a mitochondria‐targeting group and a specific peroxynitrite‐reactive trigger, was synthesized and demonstrated superior performance in cells. This strategy could lead to customization of aPSs applicable to a specific PDT.     

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.     

GSH and H2O2 Co-Activatable Mitochondria-Targeted Photodynamic Therapy under Normoxia and Hypoxia

Currently, photosensitizers (PSs) that are microenvironment responsive and hypoxia active are scarcely available and urgently desired for antitumor photodynamic therapy (PDT). Presented herein is the design of a redox stimuli activatable metal-free photosensitizer (aPS), also functioning as a pre-photosensitizer as it is converted to a PS by the mutual presence of glutathione (GSH) and hydrogen peroxide (H₂O₂) with high specificity on a basis of domino reactions on the benzothiadiazole ring. Superior to traditional PSs, the activated aPS contributed to efficient generation of reactive oxygen species including singlet oxygen and superoxide ion through both type 1 and type 2 pathways, alleviating the aerobic requirement for PDT. Equipped with a triphenylphosphine ligand for mitochondria targeting, ^mito aPS showed excellent phototoxicity to tumor cells with low light fluence under both normoxic and hypoxic conditions, after activation by intracellular GSH and H₂O₂. The ^mito aPS was also compatible to near infrared PDT with two photon excitation (800 nm) for extensive bioapplications.     

Nd3+‐Sensitized Upconversion Metal–Organic Frameworks for Mitochondria‐Targeted Amplified Photodynamic Therapy

Herein, we report the design and synthesis of a mitochondria‐specific, 808 nm NIR light‐activated photodynamic therapy (PDT) system based on the combination of metal–organic frameworks (MOFs) and upconversion photochemistry with an organelle‐targeting strategy. The system was synthesized through the growth of a porphyrinic MOF on Nd³⁺‐sensitized upconversion nanoparticles to achieve Janus nanostructures with further asymmetric functionalization of the surface of the MOF domain. The PDT nanoplatform allows for photosensitizing with 808 nm NIR light, which could effectively avoid the laser‐irradiation‐induced overheating effect. Furthermore, mitochondria‐targeting could amplify PDT efficacy through the depolarization of the mitochondrial membrane and the initiation of intrinsic apoptotic pathway. This work sheds light on the hybrid engineering of MOFs to combat their current limitations for PDT.     

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.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Effects of the Oxygenation Level on Formation of Different Reactive Oxygen Species During Photodynamic Therapy

We examined the effect of the oxygenation level on efficacy of two photosensitizing agents, both of which target lysosomes for photodamage, but via different photochemical pathways. Upon irradiation, the chlorin termed NPe6 forms singlet oxygen in high yield while the bacteriopheophorbide WST11 forms only oxygen radicals (in an aqueous environment). Photokilling efficacy by WST11 in cell culture was impaired when the atmospheric oxygen concentration was reduced from 20% to 1%, while photokilling by NPe6 was unaffected. Studies in a cell‐free system revealed that the rates of photobleaching of these agents, as a function of the oxygenation level, were correlated with results described above. Moreover, the rate of formation of oxygen radicals by either agent was more sensitive to the level of oxygenation than was singlet oxygen formation by NPe6. These data indicate that the photochemical process that leads to oxygen radical formation is more dependent on the oxygenation level than is the pathway leading to formation of singlet oxygen.