Masatoshi Beppu

Photodynamic therapy with talaporfin sodium induces dose-dependent apoptotic cell death in human glioma cell lines

OBJECTIVE To investigate the kinetics of cell death in human glioma cell lines induced by photodynamic therapy (PDT) with the second-generation photosensitizer talaporfin sodium (TS) and a 664-nm diode laser. MATERIALS AND METHODS Three human glioma cell lines (T98G, A172, U251) were studied. After incubation of the cell lines with various concentrations of TS for 4 h, PDT using diode laser irradiation at 33 mW/cm² and 10 J/cm² was performed. Cell viability and changes in cell morphology were examined by the Cell Counting Kit-8 assay and phase-contrast microscopy, respectively. In addition, to evaluate the pathology of cell death, changes in cell viability after treatment with a caspase activation inhibitor and an autophagy inhibitor were also examined. RESULTS In all 3 human glioma cell lines, TS induced dose-dependent cell death. However, the 50% lethal dose of TS varied among these cell lines. The main morphological feature of cell death was shrinkage of the cell body, and the number of cells with this morphological change increased in a time-dependent manner, resulting in cell death. In addition, a dose-dependent improvement in cell viability by the caspase inhibitor Z-VAD-fmk was observed. CONCLUSION PDT with TS induces dose-dependent apoptosis in human glioma cell lines. However, the sensitivity to PDT varied among the cell lines, indicating a possible difference in the intracellular content of TS, or a difference in the susceptibility to the intracellular oxidative stress caused by PDT.

Nucleolin is a receptor for maleylated-bovine serum albumin on macrophages.

Scavenger receptors have a broad range of functions that include pathogen clearance, and identification of the scavenger receptor family has been of great benefit to the field of physiology. The shuttling-protein nucleolin has recently been shown to possess scavenger receptor-like activity. We therefore investigated whether or not nucleolin is a receptor for maleylated-bovine serum albumin (maleylated-BSA), which is a common ligand for scavenger receptors. Binding and phagocytosis of native control-BSA by thioglycollate-elicited mouse peritoneal macrophages was weak, but that of maleylated-BSA was strong. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with maleylated-BSA but not control-BSA or maleic anhydride. Further, co-treatment of macrophages with anti-nucleolin antibody, but not control-immunoglobulin G, inhibited binding of maleylated-BSA. In addition, antineoplastic guanine rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer, inhibited binding of maleylated-BSA. Further, binding of maleylated-BSA to nucleolin-transfected HEK293 cells was higher than that by control HEK cells. These results indicate that nucleolin is a receptor that enables macrophages to recognize maleylated-BSA.

Concomitant treatment with temozolomide enhances apoptotic cell death in glioma cells induced by photodynamic therapy with talaporfin sodium

BACKGROUND Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. We have previously reported that PDT induces apoptotic cell death in glioma cells when the photosensitizer talaporfin sodium (NPe6) is used. Here, we investigated the combined effect of NPe6-PDT with temozolomide, a DNA-alkylating drug used in glioma therapy. METHODS Human glioblastoma T98G cells and human glioma U251 cells were used as glioma cells. Cell viability was evaluated by WST-8 assay. Apoptosis was evaluated by measurement of caspase-3 activity and DNA-fragmentation. Intracellular reactive oxygen species were evaluated by dihydrorhodamine assay. RESULTS While the degree of NPe6-PDT induced cell death unchanged in T98G and U251 cells when temozolomide treatment was adjuvant, it was dose-dependently increased by concomitant treatment with temozolomide. Further, concomitantly administered temozolomide dose-dependently increased caspase-3 activity and DNA-fragmentation, while adjuvant-temozolomide did not. These results are suggesting that concomitantly administered temozolomide potentiates the effect of NPe6-PDT to facilitate apoptotic cell death. Additionally, concomitantly administered temozolomide increased intracellular NPe6-fluorescence and reactive oxygen species, suggesting that the augmentation effect of combined treatment may be due to increased intracellular accumulation of NPe6. CONCLUSION These results suggest that concomitant treatment with NPe6-PDT and temozolomide is a potentially useful therapy for glioma.

Macrophage Recognition of Thiol-Group Oxidized Cells: Recognition of Carbohydrate Chains by Macrophage Surface Nucleolin as Apoptotic Cells

The mechanism was investigated for macrophage recognition of cells oxidized by diamide, a thiol group-specific oxidizing reagent. Jurkat cells exposed to various concentrations of diamide were recognized by macrophages, the cells exposed to 25 µM diamide being best recognized. CD43, a major glycoprotein on the Jurkat cell surface, tended to form clusters upon diamide oxidization, and pretreating Jurkat cells with the anti-CD43 antibody inhibited macrophage binding. This indicates that macrophages appeared to recognize CD43. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and a Western blot analysis of CD43 of the diamide-oxidized cells showed no increase in the amount of cross-linked CD43 compared with control cells, indicating that cross-linking of CD43 by a disulphide bond was not involved in the clustering. Both CD43 clustering and binding of the oxidized cells to macrophages was prevented by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), suggesting that the oxidized and macrophage-bound cells were undergoing apoptosis. A closer examination revealed that the caspase-3 activity, chromatin condensation, and DNA fragmentation in Jurkat cells were all increased by oxidation. The macrophage receptor involved in the binding appeared to be the cell-surface protein, nucleolin; an anti-nucleolin antibody treatment inhibited the binding. These results suggest that thiol group-oxidized cells underwent early apoptosis and were recognized by nucleolin on macrophages as early apoptotic cells.