Lasers Med Sci (2015) 30:1739–1745 DOI 10.1007/s10103-015-1783-9

ORIGINAL ARTICLE

Photodynamic therapy using talaporfin sodium induces concentration-dependent programmed necroptosis in human glioblastoma T98G cells Yuichi Miki 1 & Jiro Akimoto 2 & Keiko Moritake 1 & Chihiro Hironaka 1 & Yasuyuki Fujiwara 1

Received: 10 March 2015 / Accepted: 15 June 2015 / Published online: 25 June 2015 # Springer-Verlag London 2015

Abstract 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-Laspartyl 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

* Yasuyuki Fujiwara [email protected] 1

Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

2

Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku, Tokyo 160-0023, Japan

(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. Keywords Autophagy . Necroptosis . Glioma . Photodynamic therapy . Talaporfin sodium

Introduction Photodynamic therapy (PDT) is employed as a treatment methodology for several different cancers [1, 2]. Photosensitizers are selectively incorporated into neoplastic tissue and connecting vasculature, and then light irradiation induces production of reactive oxygen species, resulting in selective tumor cell death [3]. A previous study demonstrated that PDT induces several types of cell death, including apoptosis, necrosis, and autophagy, depending on the photosensitizer, PDT protocol, and tumor type [2]. These forms of cell death are not mutually exclusive, and previous evidence indicates the potential for co-occurrence [4]. Autophagy is characterized by the formation of microtubuleassociated protein 1 light-chain 3 (LC3)-II and autophagosomes. During the early stages of autophagy, LC3-I is converted to phosphatidylethanolamine-conjugated LC3-II, which subsequently forms autophagosomes [5]. Autophagosomes have enveloping membranes that sequester intracellular components, which then fuse with lysosomes to form autolysosomes, and lysosomal enzymes digest the contents. Although autophagy is associated with cytoplasmic turnover and cell survival via degradation of dysfunctional proteins and organelles in autolysosomes, excessively high autophagy levels can lead to cell death [6]. Necrosis is characterized by vacuolization of the cytoplasm and loss of plasma-membrane integrity, resulting in the release

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of cellular contents and the inflammation of surrounding tissue [7]. Necrotic cell death has been described as a passive and unorganized type of cell death. However, recent evidence suggests that necrotic cell death can be actively propagated as part of a signal transduction pathway [2]. Necroptosis has recently been suggested as a form of programmed necrosis [8, 9]. Necroptosis was first found as a signaling necrosis induced by tumor necrosis factor [8, 9]. While no unique biochemical marker for necroptosis has been identified [10], cells undergoing necroptosis display both necrotic events [e.g., lactate dehydrogenase (LDH) leakage through plasma membrane abrogation] and autophagic events (e.g., LC3-II formation and autophagosome formation) [8]. Kaczmarek et al. reported that necroptosis is dependent on the kinase activity of receptorinteracting protein kinase (RIP)-1 and RIP-3 [10], and Zhao et al. reported that mixed lineage kinase domain-like protein (MLKL) is a key RIP-3 downstream component of necroptosis [11] and MLKL is essential for Ca2+ influx, which is involved in plasma membrane rupture during necroptosis [12]. In addition to tumor necrosis factor-induced necroptosis, Coupienne et al. showed that RIP-3-dependent programmed necrosis is also observed in photosensitizer 5-aminolevulinic acid-mediated PDT-treated human glioblastoma LN18 cells [13]. These recent findings have facilitated our understanding of necrotic regulation. We previously examined the safety and efficacy of PDT using mono-L-aspartyl chlorine e6 (NPe6) (NPe6-PDT) as an additional intraoperative treatment in malignant glioma patients. Combination of NPe6-PDT with surgical resection was found to achieve better therapeutic results than conventional protocols, particularly in patients with newly diagnosed malignant glioma [14]. We also examined the effect of NPe6PDT on cell death modalities in glioma cells, with findings showing that NPe6-PDT induces mitochondrial apoptotic cell death accompanied by necrotic cell death [15]. However, despite the involvement of necrosis in glioma cell death following NPe6-PDT [15], the necrotic mechanism remains poorly understood. Determining the type of cell death and control of cell death modality in PDT treatment might be important when considering the use of PDT to effective medical treatment. 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 human glioblastoma T98G cells.

Lasers Med Sci (2015) 30:1739–1745

antibody from Medical and Biological Laboratories (Nagoya, Japan). Anti-RIP-1, anti-RIP-3, anti-MLKL, and anti-β-actin antibodies were purchased from Novus Biologicals, LLC (Littleton, CO, USA), Premo™ autophagy sensors from Invitrogen (Life Technologies Japan, Tokyo, Japan), necrostatin-1 and RPMI 1640 medium from SigmaAldrich (St. Louis, MO, USA), transfection reagent, control siRNA, RIP-1 siRNA, RIP-3 siRNA, MLKL siRNA, and siRNA reagent system from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), fetal bovine serum (FBS) from Nichirei Biosciences, Inc. (Tokyo, Japan), polyvinylidenefluoride membrane from Merck KGaA (Darmstadt, Germany), and skim milk from Wako Pure Chemical Industries (Osaka, Japan). PDT treatment of glioma cells Human glioblastoma T98G cells (Riken Cell Bank, Tsukuba, Japan) were seeded in a 96-well culture plate at 1×104 cells/ well in 10 % FBS-supplemented RPMI 1640 medium (10 % FBS-RPMI 1640) at 37 °C in 5 % CO2 atmosphere for 24 h. After incubation, the cells were washed using Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline [DPBS(-)], and incubated with 0–50 μg/ml NPe6 for 4 h in 10 % FBSRPMI 1640. After 4 h, cells were washed with DPBS(-) and incubated for another 1 h in 10 % FBS-RPMI 1640 alone. The cells were then immersed in fresh 10 % FBS-RPMI 1640 and subjected to laser irradiation [wavelength 664 nm, laser power 33 mW/cm2, total dose of laser irradiation 10 J/cm2 (irradiation time 303 s)] using a semiconductor laser irradiator (Panasonic Healthcare Co., Ltd., Ehime, Japan). Measurement of LDH activity The appearance of necrotic cell death was confirmed by leakage of LDH from the cell layer into conditioned medium. At 24 h after NPe6-PDT treatment, LDH activity in the conditioned medium was measured using an LDH cytotoxicity assay kit in accordance with the manufacturer’s instructions. Briefly, 30 μl of the conditioned medium of each sample was transferred to a 96-well plate and added 30 μl of LDH reaction solution. After 30-min incubation at room temperature, 30 μl of reaction stop solution was added and absorbance was immediately measured at 490 nm (reference wavelength 680 nm) using a microspectrometer (Varioskan Flash microplate reader; Thermo Scientific, Waltham, MA, USA), and LDH activity was evaluated by absorbance data.

Materials and methods Detection of LC3-I and LC3-II proteins in T98G cells Materials NPe6 was obtained from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan), an LDH cytotoxicity assay kit from Cayman Chemical Company (Ann Arbor, MI, USA), and anti-LC3

Twenty-four hours after NPe6-PDT treatment, T98G cells were lysed with lysis buffer [15], and cell lysate containing 1 μg of protein was analyzed by SDS-PAGE under reducing conditions. The gel was transblotted to a polyvinylidenefluoride

Lasers Med Sci (2015) 30:1739–1745

Transfection of LC3B-GFP and (G120A) LC3B-GFP to T98G cells (G120A) LC3B was used as a negative control because its LC3 glycine-to-alanine mutation (120th amino acid sequence from the N-terminus) makes the C-terminus uncleavable, and thus the mutated LC3 cannot bind to autophagosome membranes [16]. Either LC3B-GFP or (G120A) LC3B-GFP was expressed in T98G cells using Premo™ Autophagy Sensors, a commercial assay kit. Briefly, T98G cells were seeded in a 96-well culture plate at 1×104 cells/well. Either LC3B-GFP complementary DNA (cDNA) or (G120A) LC3B-GFP cDNA were then added to this plate. These cells were then incubated at 37 °C for 24 h to induce expression of each recombinant protein and subsequently subjected to NPe6-PDT. Twenty-four hours after NPe6PDT, fluorescence of intracellular recombinant proteins was observed at emission wavelength of 530 nm (exCitation wavelength 488 nm) using a using confocal laser-scanning fluorescence microscopy (FV1000D; Olympus, Tokyo, Japan). siRNA treatment and detection of RIP-1, RIP-3, and MLKL in T98G cells Control short interfering RNA (siRNA), RIP-1 siRNA, RIP-3 siRNA, and MLKL siRNA were transfected to T98G cells via siRNA transfection using a commercial assay kit (siRNA reagent system). Briefly, T98G cells were seeded at 1×104 cells/ well with transfection reagent and 60 nM of each siRNA in transfection medium (Santa Cruz Biotechnology). Five hours after seeding, double concentrated 20 % FBS-supplemented RPMI 1640 medium was added to each seeded plate of cells. Another 19 h later, siRNA-transfected cells were treated with NPe6-PDT, as described above. Separately, siRNAtransfected cells were lysed with a lysis buffer [15], and knockdown effects of RIP-1, RIP-3, and MLKL were confirmed by Western blot analysis and densitometric analysis using ImageJ. Statistical analysis Data were represented as mean values with standard deviations (mean±SD). Differences between treatments were determined using Student’s t test, with p values of

Photodynamic therapy using talaporfin sodium induces concentration-dependent programmed necroptosis in human glioblastoma T98G cells.

Photodynamic therapy (PDT) using photosensitizer induces several types of cell death, such as apoptosis, necrosis, and autophagy, depending on the PDT...
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