Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts

Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin has been approved by the U.S. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc-OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR-3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR-3 tumors (approximately 80-130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (lambda = 672 nm, 150 J/cm2, 150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post-PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post-PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post-PDT. Western blotting showed the cleavage of the 116 kDa native poly(ADP-ribose) polymerase (PARP) into fragments of approximately 90 kDa, another indication of apoptosis, and the presence of p21/WAF1/CIP1 (p21) in all PDT-treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4-PDT response. Clinical trials of Pc 4-PDT are warranted.     

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.     

Lutetium Texaphyrin (PCI-0123): A Near-Infrared, Water-Soluble Photosensitizer

Lutetium texaphyrin, PCI-0123, is a pure, water-soluble photosensitizer with a large broad absorption band centered at 732 nm. The compound was tested for photodynamic therapy (PDT) effectiveness in a murine mammary cancer model. The texaphyrin macrocycle as illustrated by magnetic resonance imaging and ¹⁴C-radiolabeled texaphyrin studies was shown to be tumor selective; a tumor-to-muscle ratio of 10.55 was seen after 5 h. Lutetium texaphyrin, at a drug dose of 20 μmol/kg with irradiation 5 h postinjection at 150 J/cm² and 150 mW/cm², had significant efficacy (P < 0.0001) in treating neoplasms of moderate size (40 ± 14 mm³) and also had significant efficacy ( P < 0.0001) in treating larger neoplasms (147 ± 65 mm³). The PDT efficacy was correlated with the time interval between PCI-0123 administration and light exposure. A 100% cure rate was achieved when photoirradiation took place 3 h postinjection compared to 50% for 5 h using 10 μmol/kg and 150 J/cm² at 150 mW/cm². The PDT efficacy was attributable to the selective uptakehetention of the texaphyrin photosensitizer in addition to the depth of light penetration achievable at the 732 nm laser irradiation.     

Apoptosis is an Early Event During Phthalocyanine Photodynamic Therapy-Induced Ablation of Chemically Induced Squamous Papillomas in Mouse Skin

Abstract— Photodynamic therapy (PDT) is a promising new modality to treat malignant neoplasms including superficial skin cancers. In our search for an ideal photosensitizer for PDT, Pc 4, a silicon phthalocyanine, has shown promising results both in in vitro assays and in implanted tumors. In this study we assessed the efficacy of Pc 4 PDT in the ablation of murine skin tumors; and the evidence for apoptosis during tumor ablation was also obtained. The Pc 4 was administered through tail vein injection to SENCAR mice bearing chemically induced squamous papillomas, and 24 h later the lesions were illuminated with an argon ion-pumped dye laser tuned at 675 nm for a total light dose of 135 J/cm². Within 72-96 h, almost complete tumor shrinkage occurred; no tumor regrowth was observed up to 90 days post-PDT. As evident by nucleosome-size DNA fragmentation, appearance of apoptotic bodies in hematoxylin and eosin staining and direct immunoperoxidase detection of digoxigenin-labeled genomic DNA in sections, apoptosis was clearly evident 6 h post-PDT at which time tumor shrinkage was less than 30%. The apoptotic bodies, as evident by the condensation of chromatin material around the periphery of the nucleus and increased vacuolization of the cytoplasm, were also observed in electron microscopic studies of the tumor tissues following Pc 4 PDT. The extent of apoptosis was greater at 15 h than at 6 and 10 h post-PDT. Taken together, our results clearly show that Pc 4 may be an effective photosensitizer for PDT of nonmelanoma skin cancer, and that apoptosis is an early event during this process.     

Irradiation-induced enhancement of Pc 4 fluorescence and changes in light scattering are potential dosimeters for Pc 4-PDT

Phthalocyanine 4 (Pc 4) is a promising photosensitizer currently in clinical trials. Photobiological responses to Pc 4 photodynamic therapy (Pc 4-PDT) have been characterized extensively, but relatively little has been done to evaluate dose metrics for this sensitizer. We describe an irradiation-induced increase in fluorescence in tumor cell monolayers. This increase is due solely to enhanced fluorescence from Pc 4, as confirmed by confocal spectroscopy. In EMT6 cells incubated with 250 nM Pc 4 for 24 h, the maximum increase in fluorescence is approximately 3.7-fold above baseline levels. This increase occurs over a range of fluences, 0.05-0.6 J cm(-2), where clonogenic survival decreases by 3 orders of magnitude. Light scattering measurements performed on similarly treated EMT6 cells in suspension suggested a Pc 4-PDT-mediated mitochondrial swelling of approximately 13% at 0.6 J cm(-2), where fluorescence enhancement saturates under these treatment conditions. Fluorescence imaging and light scattering experiments performed at a five-fold lower Pc 4 incubation concentration revealed a reduced fluorescence enhancement at a five-fold higher fluence, which produced comparable mitochondrial swelling. Taken together, these data suggest that Pc 4 is initially aggregated at high local concentration in mitochondria and that irradiation relaxes the quenching of Pc 4 fluorescence through a mechanism that may involve mitochondrial swelling.     

Fluence Rate-Dependent Photobleaching of Intratumorally Administered Pc 4 Does not Predict Tumor Growth Delay

We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg kg^?1 Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW cm^?2 to a fluence of 100 J cm^?2. While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared with drug‐only controls. There was no significant difference in tumor responses to these two irradiances (P = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW cm^?2 group, enhanced photobleaching was associated with prolonged growth delay (P = 0.188), while at 150 mW cm^?2 this trend was reversed (P = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc 4‐PDT under these treatment conditions.     

Photodynamic Therapy with a Diode Laser for Implanted Fibrosarcoma in Mice Employing Mono-L-Aspartyl Chlorin E6

Abstract— The authors performed photodynamic therapy (PDT), avoiding any hyperthermic effects, using a newly developed diode laser and photosensitizer, mono-L-aspar-tyl chlorin e₆ (NPe6), of Meth-A fibrosarcoma implanted in mice and achieved tumor therapeutic benefit. The photodynamic light treatment was performed 5 h following the photosensitizer administration. With 5.0 mg/kg NPe6 and light doses of 50, 100, 150 and 200 J/cm², the tumor cure rates were 20, 50, 70 and 90%, respectively. With 100 J/cm² laser exposure and NPe6 doses of 1.25, 2.5, 5.0, 7.5 and 10.0 mg/kg, the tumor cure rates were 0, 20, 50, 70 and 90%, respectively. A charge-coupled device (CCD) camera system was employed to measure the NPe6 fluorescence intensity correlating with the residual amount of the photosensitizer at deferent depth from the tumor surface. The ratios of the NPe6 fluorescence intensity at 3 mm from the tumor surface following 50, 100, 150 and 200 J/cm² laser exposure to no laser exposure were 0.73, 0.36, 0.22 and 0.16, respectively. With samples sectioned at 1 mm depth, after 50 J/cm² and the same photosensitizer dose (5 mg/kg) this ratio was 0.19. These results suggest that a certain increase in the tumor tissue level of NPe6 and a certain increase of laser light dose reaching deeper layers of tumor caused an increase in percent cure. In addition, the effectiveness of PDT depends on the total laser dose reaching deeper layers of tumors. Furthermore, the effectiveness of PDT tends to correlate with the amount of NPe6 photobleaching by PDT.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

Exploitation of Immune Response-eliciting Properties of Hypocrellin Photosensitizer SL052-based Photodynamic Therapy for Eradication of Malignant Tumors

A diaminophenyl derivative of hypocrellin B (SL052) has been developed as a photosensitizer for use in photodynamic therapy (PDT) of solid tumors. Testing SL052-PDT on mouse carcinoma and fibrosarcoma models revealed a typical response seen with clinically established photosensitizers featuring initial rapid tumor ablation with ensuing recurrence at rates dependent on photosensitizer/light doses. Elevated numbers of immune cells were found in lymph nodes draining SCCVII mouse squamous cell carcinomas treated by SL052-PDT (in particular T cells), and the accumulation of degranulating cytotoxic T cells was detected at the tumor-treated site. This indicates that a significant contribution to tumor cures is elicited by an antitumor adaptive immune response. Two different immunotherapy agents, γ-interferon and antibody blocking inhibitory FcγRIIB receptor, were both found to be highly effective in potentiating the curative effect of SL052-PDT with SCCVII tumors. Combining SL052-PDT with FcγRIIB-blocking antibody treatment caused a further increase in the number of cells in tumor-draining lymph nodes and in degranulating CD8⁺ cells, suggesting the amplification of the immune response induced by PDT. Vaccines consisting of SCCVII cells treated with SL052-PDT in vitro were effective in reducing growth of established subcutaneous SCCVII tumors. In conclusion, PDT mediated by SL052 is suitable to be integrated with various immunotherapy protocols.     

The immunosuppressive effects of phthalocyanine photodynamic therapy in mice are mediated by CD4+ and CD8+ T cells and can be adoptively transferred to naive recipients

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors but it is also immunosuppressive which may reduce its therapeutic efficacy. The purpose of our study was to elucidate the role of CD4+ and CD8+ T cells in PDT immunosuppression. Using silicon phthalocyanine 4 (Pc4) as photosensitizer, nontumor-bearing CD4 knockout (CD4-/-) mice and their wild type (WT) counterparts were subjected to Pc4-PDT in a manner identical to that used for tumor regression (1 cm spot size, 0.5 mg kg(-1) Pc4, 110 J cm(-2) light) to assess the effect of Pc4-PDT on cell-mediated immunity. There was a decrease in immunosuppression in CD4-/- mice compared with WT mice. We next examined the role of CD8+ T cells in Pc4-PDT-induced immunosuppression using CD8-/- mice following the same treatment regimen used for CD4-/- mice. Similar to CD4-/- mice, CD8-/- mice exhibited less immunosuppression than WT mice. Pc4-PDT-induced immunosuppression could be adoptively transferred with spleen cells from Pc4-PDT treated donor mice to syngenic naive recipients (P < 0.05) and was mediated primarily by T cells, although macrophages were also found to play a role. Procedures that limit PDT-induced immunosuppression but do not affect PDT-induced regression of tumors may prove superior to PDT alone in promoting long-term antitumor responses.     

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

In vitro and in vivo evaluation of improved EGFR targeting peptide-conjugated phthalocyanine photosensitizers for tumor photodynamic therapy

A serial of peptide-conjugated zinc phthalocyanines with finely tuned structure modification were prepared and one optimized conjugate showed improved targeting towards tumors and abolished inoculated tumors with only a single PDT treatment in a subcutaneous xenograft tumor model, making this approach a promising therapeutic agent for the treatment of cancer.     

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells vs A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.     

A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but Not a Mitochondrion-targeted Photosensitizer

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid^−/− cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.     

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.     

Amelioration of Airway Stenosis in Rabbit Models by Photodynamic Therapy with Talaporfin Sodium (NPe6)

It is difficult to treat patients with acquired airway stenosis, and the quality of life of such patients is therefore lowered. We have suggested the application of photodynamic therapy (PDT) as a new treatment for airway stenosis and have determined the efficacy of PDT in animal disease models using a second-generation photosensitizer with reduced photosensitivity. An airway stenosis rabbit model induced by scraping of the tracheal mucosa was administered NPe6 (5 mg kg⁻¹), and the stenotic lesion was irradiated with 670 nm light emitted from a cylindrical diffuser tip at 60 J cm⁻² under bronchoscopic monitoring. PDT using NPe6 improved airway stenosis ( P  = 0.043) and respiratory stridor. A significant prolongation of survival time was seen in the PDT-treated animals compared to that in the untreated animals ( P  = 0.025) and 44% of the treated animals achieved long-term survival (>60 days). In conclusion, PDT using NPe6 is effective for improvement in airway stenosis.     

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.     

Promotion of photodynamic therapy-induced apoptosis by the mitochondrial protein Smac/DIABLO: dependence on Bax

Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO-GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO-GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33,342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c-dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO-GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.     

Photochemical Internalization of Bleomycin is Superior to Photodynamic Therapy Due to the Therapeutic Effect in the Tumor Periphery

Photochemical internalization (PCI) is under development for clinical use in treatment of soft tissue sarcomas and other solid tumors. PCI may release endocytosed bleomycin (BLM) into the cytosol by photochemical rupture of the endocytic vesicles. In this study, the human fibrosarcoma xenograft HT1080 was transplanted into the leg muscle of athymic mice. The photosensitizer disulfonated aluminum phthalocyanine (AlPcS_2a) and BLM were systemically administrated 48 h and 30 min, respectively, prior to light exposure at 670 nm (30 J cm⁻²). The purposes of this study were to evaluate the treatment response to AlPcS_2a-photodynamic therapy (PDT) and AlPcS_2a-PDT in combination with BLM ( i.e. PCI of BLM) in an orthotopic, invasive and clinically relevant tumor model and to explore the underlying response mechanisms caused by PDT and PCI of BLM. The treatment response was evaluated by measuring tumor growth, contrast-enhanced magnetic resonance imaging (CE-MRI), histology and fluorescence microscopy. The results show that PCI of BLM is superior to PDT in inducing tumor growth retardation and acts synergistically as compared to the individual treatment modalities. The CE-MRI analyses 2 h after AlPcS_2a-PDT and PCI of BLM identified a treatment-induced nonperfused central zone of the tumor and a well-perfused peripheral zone. While there were no differences in the vascular response between PDT and PCI, the histological analyses showed that PDT caused necrosis in the tumor center and viable tumor cells were found in the tumor periphery. PCI caused larger necrotic areas and the regrowth in the peripheral zone was almost completely inhibited after PCI. The results indicate that PDT is less efficient in the tumor periphery than in the tumor center and that the treatment effect of PCI is superior to PDT in the tumor periphery.