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     

Epigenetically Enhanced Photodynamic Therapy (ePDT) is Superior to Conventional Photodynamic Therapy for Inducing Apoptosis in Cutaneous T‐Cell Lymphoma

Conventional photodynamic therapy with aminolevulinate (ALA‐PDT) selectively induces apoptosis in diseased cells and is highly effective for treating actinic keratoses. However, similar results are achieved only in a subset of patients with cutaneous T‐cell lymphoma (CTCL). Our previous work shows that the apoptotic resistance of CTCL correlates with low expression of death receptors like Fas cell surface death receptor (FAS), and that methotrexate upregulates FAS by inhibiting the methylation of its promoter, acting as an epigenetic derepressor that restores the susceptibility of FAS‐low CTCL to caspase‐8‐mediated apoptosis. Here, we demonstrate that methotrexate increases the response of CTCL to ALA‐PDT, a concept we refer to as epigenetically enhanced PDT (ePDT). Multiple CTCL cell lines were subjected to conventional PDT versus ePDT. Apoptotic biomarkers were analyzed in situ with multispectral imaging analysis of immunostained cells, a method that is quantitative and 5× more sensitive than standard immunohistology for antigen detection. Compared to conventional PDT or methotrexate alone, ePDT led to significantly greater cell death in all CTCL cell lines tested by inducing greater activation of caspase‐8‐mediated extrinsic apoptosis. Upregulation of FAS and/or tumor necrosis factor‐related apoptosis‐inducing ligand pathway components was observed in different CTCL cell lines. These findings provide a rationale for clinical trials of ePDT for CTCL.     

基于光敏剂的光动力疗法

光动力治疗日益受到关注。本文综述了光敏剂在光动力疗法领域中的研究进展,包括它的性能改进、作用机理和靶点。并对光动力疗法的发展前景进行了展望。     

Delayed Oxidative Photodamage induced by Photodynamic Therapy

Abstract— Apoptotic DNA fragmentation was observed 60 min after photodynamic therapy of murine leukemia cells in culture, using either of two photosensitizers with predominantly lysosomal targets. When the radical scavengers trolox or α-tocopherol succinate were present during irradiation, the subsequent appearance of apoptotic cells was prevented, as was phototoxicity. Addition of either scavenger during the 60 min after irradiation provided only partial protection from apoptosis and phototoxicity; this protection was abolished if the addition was delayed for 10 min. These results are consistent with a model whereby long-persisting photoproducts continue the initiation of apoptosis for approximately 10 min after irradiation has ceased.     

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.     

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.     

Therapeutic Enhancement of Verteporfin-mediated Photodynamic Therapy by mTOR Inhibitors

Photodynamic therapy (PDT) with photosensitizer verteporfin is a clinically approved vascular disrupting modality that is currently in clinical trial for cancer treatment. In this study, we evaluated PDT in combination with either mTORC1 inhibitor rapamycin or mTORC1/C2 dual inhibitor AZD2014 for therapeutic enhancement in SVEC endothelial cells. Verteporfin-PDT alone induced cell apoptosis by activating the intrinsic apoptotic pathway. However, it increased the expression of anti-apoptotic protein MCL-1 and the phosphorylation of S6, a downstream molecule of mTOR signaling. In contrast, mTOR inhibitors rapamycin and AZD2014 did not induce apoptosis in SVEC cells. They suppressed MCL-1 expression and S6 phosphorylation and imposed a potent inhibition on cell proliferation. PDT in combination with mTOR inhibitors activated the intrinsic apoptotic pathway and resulted in increased apoptosis. Combination treatments also led to sustained inhibition of cell proliferation. Although AZD2014 was more effective for cell growth inhibition and PDT enhancement than rapamycin at the higher concentrations examined in the study, both inhibitors effectively enhanced PDT response, suggesting that inhibition of mTORC1 is crucial for PDT enhancement. Our results indicate that mTOR inhibitors mechanistically cooperate with PDT for enhanced cell death and sustained growth inhibition, supporting a combination approach for therapeutic enhancement.     

Deletion of Alloantigen-Activated Cells by Aminolevulinic Acid-Based Photodynamic Therapy

Protoporphyrin IX (PpIX), an endogenously synthesized photosensitizer, can transiently accumulate in activated lymphocytes following administration of the heme precursor 5-aminolevulinic acid (ALA). One possible mechanism of this in lymphocyte accumulation is that actively dividing cells use intracellular iron stores for cytochrome and DNA synthesis and thus do not inactivate PpIX, the photoactive precursor of heme, by iron incorporation. This selective accumulation in activated cells should allow targeting by photodynamic therapy (PDT). To determine the effect of this accumulation, we studied PDT effects on the in vitro correlate of transplantation rejection: the one-way mixed lymphocyte reaction (MLR). Selective phototoxicity was determined by photoirradiating ALA-treated, MLR-activated cells and measuring subsequent stimulation either in a secondary MLR or with phytohemagglutinin (PHA). We found that proliferation of MLR-activated lymphocytes incubated with ALA and treated with light was only 12-20% of controls (ALA+, no light) after rechallenge with the stimulator cells (P < 0.05), although their response to nonspecific PHA stimulation was similar to controls. Thus alloantigen-specific depletion was shown. The data suggest a role for ALA-PDT in the treatment of diseases that require the selective elimination of activated lymphocytes and possibly as an immunomodulator.     

Photodynamic Therapy for Gastrointestinal Cancer

The clinical application of photodynamic therapy (PDT) for gastrointestinal (GI) neoplastic lesions has been developed with appreciation for the great efforts and kind support of Dr. Tom Dougherty and his followers’ contributions. There are several published studies on clinical PDT in the field of GI oncology. Esophageal cancer was one of the first clinical indications for PDT that was approved as an endoscopic procedure in both the United States and Japan. PDT was initially used as a palliative local treatment for patients with obstructive esophageal cancer. PDT is also indicated for eradicative therapy for dysplastic Barret’s esophagus, which is the precursor state of esophageal adenocarcinoma, with the support of level one evidence. In Japan, PDT was approved as a curative treatment for superficial esophageal carcinoma lesions, which are difficult to treat with endoscopic resection. Further, PDT using second-generation photosensitizers is approved for early local failure after radiotherapy, for which treatment with other modalities is difficult. PDT has also been assessed in other GI cancers, including gastric cancer, biliary cancer and pancreatic cancer. In this review, we overview the history and state of PDT for GI cancer.     

Techniques to Improve Photodynamic Therapy

Photodynamic therapy [dye-light therapy] is an excellent technique for use in detection and treatment of cancerous tissues. While this therapy is effective, it is limited by the phototoxic reactions that can occur in the surrounding normal tissues. These damaging side effects are of particular importance when treating neurosensory organs, such as the human eye. We report here new treatment strategies to enhance photodynamic effectiveness while limiting side effects to normal tissues.     

Enhancement of Photodynamic Cancer Therapy by Physical and Chemical Factors

The viable use of photodynamic therapy (PDT) in cancer therapy has never been fully realized because of its undesirable effects on healthy tissues. Herein we summarize some physicochemical factors that can make PDT a more viable and effective option to provide future oncological patients with better‐quality treatment options. These physicochemical factors include light sources, photosensitizer (PS) carriers, microwaves, electric fields, magnetic fields, and ultrasound. This Review is meant to provide current information pertaining to PDT use, including a discussion of in vitro and in vivo studies. Emphasis is placed on the physicochemical factors and their potential benefits in overcoming the difficulty in transitioning PDT into the medical field. Many advanced techniques, such as employing X‐rays as a light source, using nanoparticle‐loaded stem cells and bacteriophage bio‐nanowires as a photosensitizer carrier, as well as integration with immunotherapy, are among the future directions.     

A Tumor-Targeting Dual-Stimuli-Activatable Photodynamic Molecular Beacon for Precise Photodynamic Therapy

A multifunctional photodynamic molecular beacon (PMB) has been designed and synthesized which contains an epidermal growth factor receptor (EGFR)-targeting cyclic peptide and a trimeric phthalocyanine skeleton in which the three zinc(II) phthalocyanine units are each substituted with a glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS) quencher and are linked via two cathepsin B-cleavable GFLG peptide chains. This tailor-made conjugate is fully quenched in the native form due to the photoinduced electron transfer effect of the DNBS moieties and the self-quenching of the phthalocyanine units. It can target the EGFR overexpressed in cancer cells, and after receptor-mediated endocytosis, it can be activated selectively by the co-existence of intracellular GSH and cathepsin B, both of which are also overproduced in cancer cells, in terms of fluorescence emission and singlet oxygen generation. The cell-selective behavior of this PMB has been demonstrated using a range of cancer cells with different expression levels of EGFR, while the stimuli-responsive properties have been studied both in vitro and in various aqueous media. The overall results show that this advanced PMB, which exhibits several levels of control of the tumor specificity, is a promising photosensitizer for precise antitumoral photodynamic therapy.     

Photosynthetic Tumor Oxygenation by Photosensitizer-Containing Cyanobacteria for Enhanced Photodynamic Therapy

Sustained tumor oxygenation is of critical importance during type-II photodynamic therapy (PDT), which depends on the intratumoral oxygen level for the generation of reactive oxygen species. Herein, the modification of photosynthetic cyanobacteria with the photosensitizer chlorin e6 (ce6) to form ce6-integrated photosensitive cells, termed ceCyan, is reported. Upon 660 nm laser irradiation, sustained photosynthetic O₂ evolution by the cyanobacteria and the immediate generation of reactive singlet oxygen species (¹O₂) by the integrated photosensitizer could be almost simultaneously achieved for tumor therapy using type-II PDT both in vitro and in vivo. This work contributes a conceptual while practical paradigm for biocompatible and effective PDT using hybrid microorganisms, displaying a bright future in clinical PDT by microbiotic nanomedicine.     

酞菁在光动力治疗中的应用

光动力治疗因具有低毒、副作用小、抗癌广谱、高选择性等优势,正吸引着人们越来越多的关注。提高光敏剂的选择性和光毒性已经成为研究的热点。本文简单介绍了光敏剂的发展历程,并对酞菁类第三代光动力治疗光敏剂的最新研究进展进行了论述。     

酞菁在光动力治疗中的应用

光动力治疗因具有低毒、副作用小、抗癌广谱、高选择性等优势, 正吸引着人们越来越多的关注。提高光敏剂的选择性和光毒性已经成为研究的热点。本文简单介绍了光敏剂的发展历程, 并对酞菁类第三代光动力治疗光敏剂的最新研究进展进行了论述。     

Nanobody-Ferritin Conjugate for Targeted Photodynamic Therapy

Ferritin is an iron-storage protein nanocage that is assembled from 24 subunits. The hollow cavity of ferritin enables its encapsulation of various therapeutic agents; therefore, ferritin has been intensively investigated for drug delivery. The use of antibody-ferritin conjugates provides an effective approach for targeted drug delivery. However, the complicated preparation and limited protein stability hamper wide applications of this system. Herein, we designed a novel nanobody-ferritin platform (Nb-Ftn) for targeted drug delivery. The site-specific conjugation between nanobody and ferritin is achieved by transglutaminase-catalyzed protein ligation. This ligation strategy allows the Nb conjugation after drug loading in ferritin, which avoids deactivation of the nanobody under the harsh pH environment required for drug encapsulation. To verify the tumor targeting of this Nb-Ftn platform, a photodynamic reagent, manganese phthalocyanine (MnPc), was loaded into the ferritin cavity, and an anti-EGFR nanobody was conjugated to the surface of the ferritin. The ferritin nanocage can encapsulate about 82 MnPc molecules. This MnPc@Nb-Ftn conjugate can be efficiently internalized by EGFR positive A431 cancer cells, but not by EGFR negative MCF-7 cells. Upon 730 nm laser irradiation, MnPc@Nb-Ftn selectively killed EGFR positive A431 cells by generating reactive oxygen species (ROS), whereas no obvious damage was observed on MCF-7 cells. Given that ferritin can be used for encapsulation of various therapeutic agents, this work provides a strategy for facile construction of nanobody-ferritin for targeted drug delivery.     

光动力疗法用糖基光敏剂的研究进展

作为光动力疗法中至关重要的决定性因素,光敏剂的研究受到越来越多的重视.而糖基的引入,可以大大提高光敏剂母体的膜透过性和特异吸收性.从糖基光敏剂的母体结构、糖基光敏剂分子的构效关系、糖基的作用机理以及糖基光敏剂的药物动力学和代谢产物这四个方面对近年来糖基光敏剂的研究现状进行了综述,对其发展趋势进行了展望.