talaporfin and Necrosis

Photodynamic therapy (PDT) using photosensitizer induces several types of cell death, such as apoptosis, necrosis, and autophagy, depending on the PDT procedure, photosensitizer type, and cell type. We previously demonstrated that PDT using the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6; NPe6-PDT) induces both mitochondrial apoptotic and necrotic cell death in human glioblastoma T98G cells. However, details regarding the mechanism of necrosis caused by NPe6-PDT are unclear. Here, we investigated whether or not necroptosis, a recently suggested form of programmed necrosis, is involved in the necrotic cell death of NPe6-PDT-treated T98G cells. Leakage of lactate dehydrogenase (LDH) from the cell layer into conditioned medium was significantly increased by NPe6 (25 and 50 μg/ml)-PDT, indicating that NPe6-PDT induces necrosis in these cells. NPe6 (25 μg/ml)-PDT treatment also induced conversion of microtubule-associated protein 1 light-chain 3 (LC3)-I into phosphatidylethanolamine-conjugated LC3-II accompanying autophagosome formation, indicators of autophagy; however, of note, NPe6 (50 μg/ml)-PDT did not induce such autophagic changes. In addition, both necrostatin-1 (a necroptosis inhibitor) and knockdown of necroptotic pathway-related proteins [e.g., receptor interacting serine-threonine kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] inhibited leakage of LDH caused by NPe6 (25 μg/ml)-PDT. Taken together, the present findings revealed that NPe6-PDT-induced necrotic cell death is mediated in part by the necroptosis pathway in glioblastoma T98G cells.

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Chlorophyllides; Glioblastoma; Humans; L-Lactate Dehydrogenase; Microtubule-Associated Proteins; Necrosis; Phagosomes; Photochemotherapy; Photosensitizing Agents; Porphyrins

An application of photodynamic therapy for myocardial ablation, which would induce myocardial electrical conduction block, is proposed. For the proposed application, an extracellular photosensitization reaction (PR) is performed while photosensitizer is distributed in myocardial interstitial space by employing a short drug-light interval. Because the myocardial necrosis depth must be accurately controlled to prevent surrounding tissue injury during the myocardial ablation procedure, the necrosis depth during PR needs to be predicted. The purpose of this study is to investigate the availability of predicting PR-induced myocardial necrosis depth (d(nec)) using a defined fluorescence-fall amount (FA), which is the calculated result of fluorescence intensity decrease from the start of the PR multiplied by irradiation duration and corresponds to photosensitizer consumption amount under an assumption that the photosensitizer consumption rate is faster than the photosensitizer supply rate. The correlation between FA and d nec was experimentally investigated in vivo using an open-chested canine heart model with 2.5 and 5.0 mg/kg of talaporfin sodium at an irradiance of 5-20 W/cm(2) for 5-20 s. The fluorescence measurement was performed at a wavelength of 710 nm during the PR to derive FA. One week after the PR, a uniform necrosis depth was measured histopathologically as d(mnec). A logarithmic correlation between d(mnec) and FA was confirmed with R(2) = 0.69-0.80 and a d(mnec) range of 0.2-7.1 mm. The defined FA might be useful for predicting d nec for the extracellular PR in myocardium when using talaporfin sodium.

Topics: Animals; Dogs; Extracellular Space; Myocardium; Necrosis; Photochemotherapy; Photosensitizing Agents; Porphyrins; Spectrometry, Fluorescence

We proposed a new non-thermal treatment for tachyarrhythmia that employs an extracellular photosensitization reaction. Oxygen depletion may easily occur in in vitro studies of this reaction because the photosensitizing agent is often highly concentrated in such studies. The aim of the current study was to examine the progress of the extracellular photosensitization reaction and the photocytotoxicity of extracellular Talaporfin sodium on myocardial cells for application in tachyarrhythmia therapy.. Photosensitization reactions were performed in single wells of 96-well plates; Talaporfin sodium solution concentrations from 5 to 40 µg/ml, radiant exposures up to 40 J/cm(2) , and irradiance of 0.29 W/cm(2) from a continuous wave (CW) red diode laser (wavelength: 663 nm) were used. We measured transient changes of temperature, photosensitizer fluorescence, dissolved oxygen pressure, and photosensitizer solution absorbance to monitor the progress of the photosensitization reaction in the system during laser irradiation. Rat myocardial cells were cultured in 96-well plates, and the drug-light interval was set to 15 minutes. We used a WST assay to measure cell lethality 2 hours after laser irradiation.. A strong photosensitization reaction occurred several seconds after initiation of laser irradiation; this initial reaction depended upon dissolved oxygen. A gentler continuous photosensitization reaction followed the initial reaction, and was associated with temperature increases of less than 10°C. The oxygen pressure was kept in approximately 40 mmHg of the myocardial tissue oxygen pressure in the gentle photosensitization reaction phase. At radiant exposures from 10 to 40 J/cm(2) , a photosensitizer concentration of approximately 15 µg/ml was the threshold for myocardial cell necrosis in this in vitro system. The dependencies of photocytotoxicity on radiant exposure were separated into two distinct groups based on the molecular density ratio between Talaporfin sodium and albumin.. This in vitro system for the extracellular photosensitization reaction may reflect the situation in live myocardial tissue. We found that the extracellular photosensitization reaction progressed in two distinctive phases; the first phase depended upon dissolved oxygen, and the second upon the molar density ratio between Talaporfin sodium and albumin. Cell lethality due to the extracellular photosensitization reaction was influenced by both of these factors in our in vitro system. We suggest that a photosensitizer concentration of 25 µg/ml might be necessary to treat myocardial tissue with therapies involving the extracellular photosensitization reaction.

Topics: Animals; Biomarkers; Cells, Cultured; Dose-Response Relationship, Drug; Fluorescence; Lasers, Semiconductor; Myocytes, Cardiac; Necrosis; Oxygen; Photochemotherapy; Photosensitizing Agents; Porphyrins; Rats; Tachycardia

Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.

Topics: Antineoplastic Agents; Brain Neoplasms; Caspase 3; Cell Death; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Glioma; Humans; Mitochondria; Necrosis; Photochemotherapy; Photosensitizing Agents; Porphyrins

In this study, we investigated the induction of apoptosis by ultrasound in the presence of a photochemically active chlorin, mono-l-aspartyl chlorin e6 (NPe6). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of NPe6, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 80 microM NPe6 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound alone, at the same intensity, or NPe6 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and NPe6 but not in cells treated with ultrasound or NPe6 alone. In addition, NPe6 substantially enhanced nitroxide generation by ultrasound in the same acoustical arrangement. Sonodynamically-induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results suggest that the combination of ultrasound and NPe6 sonochemically induces apoptosis as well as necrosis in HL-60 cells. They further suggest that some ultrasonically-generated active species, deactivatable by histidine, are the major mediators to induce the observed apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Enzyme Activation; Free Radical Scavengers; HL-60 Cells; Humans; Necrosis; Nitrogen Oxides; Photosensitizing Agents; Porphyrins; Reactive Oxygen Species; Ultrasonic Therapy

Mono-L-aspartyl chlorin e6 (NPe6) is an effective photosensitizer with a major absorption band at 664 nm. NPe6 is potentially exploitable for photodynamic therapy (PDT) and does not cause the side effect of prolonged normal skin photosensitization. However, there are no clinical and experimental reports of its use in oral cancer till now. In the present study, we examined the effectiveness of NPe6-induced PDT with a diode laser for treatment of tongue cancer in the nude mouse. Six nude mice with experimental tongue cancer (HSC-3) were given 10 mg/kg NPe6 intravenously. Two hours later PDT was performed using a laser diode at a light dose of 100 J/cm2 and wavelength of 664 nm. Histological changes in the tumors were examined 42-72 h after PDT. Almost all of the tumors developed necrosis, while viable-like neoplastic cells remained mainly in the peripheral region of the tumor in some cases. The mean depth of necrosis below the surface was 2.1 mm. The mean tumor thickness below the surface was 2.3 mm. Tumor thickness coincided with the depth of necrosis. NPe6-induced PDT exhibited tumor selectivity and can effectively cause necrosis of tongue cancers. This therapy could be suggested for treatment of other superficial oral cancer.

Topics: Animals; Carcinoma, Squamous Cell; Female; Laser Therapy; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Photochemotherapy; Photosensitizing Agents; Porphyrins; Tongue Neoplasms

We studied the detection of apoptosis of malignant human salivary gland tumor cells induced by photodynamic therapy (PDT) using the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) in vitro and in vivo in mice receiving transplanted human salivary gland tumor (HSG) cells. An immunohistocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin (CK) 18 by activated caspase, was applied to detect the apoptosis of HSG cells induced by PDT. Significant amounts of immunoreactive products were observed in the cytoplasm of HSG cells after PDT. In vitro, M30-positive cells increased from 2 h after PDT, increased rapidly from 8 h and reached a peak 24 h after PDT. In vivo, a peak of early apoptosis was confirmed two hours after PDT. In comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, the destroyed tumor cells were observed sporadically 24 h after PDT. These results suggest that immunohistocytochemical staining with the MoAb M30 may be useful for detecting early apoptosis induced by PDT. Futhermore, PDT using NPe6 is effective in inducing apoptosis of HSG cells at an early stage, which suggests the possibility of the therapy being ideal for treatment of human malignant neoplasms.

Topics: Animals; Apoptosis; Cell Line, Tumor; Disease Models, Animal; DNA, Neoplasm; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Photochemotherapy; Photosensitizing Agents; Porphyrins; Salivary Gland Neoplasms