The study was conducted to clarify the cytocidal effect of combination therapy consisting of administration of acridine orange (AO), which is a photosensitizer, and radiation therapy using in vitro and in vivo mouse osteosarcoma models. The results revealed that AO combined with low-dose X-ray irradiation of about 1–5 Gy had a strong cytocidal effect on the cultured mouse osteosarcoma cells regardless of their chemosensitivity, and that this combination therapy inhibited growth of the in vivo mouse osteosarcoma by induction of tumor necrosis. This effect was inhibited by L-histidine, but not by mannitol. These findings suggested that AO might be excited by X-rays and kill osteosarcoma cells through the release of singlet oxygen, which is toxic to living cells. This mechanism is similar to that of photodynamic therapy with AO.

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS/ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS/ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long-or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Key words: Multidrug resistance -Osteosarcoma -Acridine orange -Photodynamic therapyThere is no doubt that chemotherapy is the most important treatment for improving the prognosis of patients with osteosarcoma. However, about 30% of these patients have been found to be resistant to chemotherapy.1-3) Therefore, overcoming multidrug resistance (MDR) is an urgent issue in the management of osteosarcomas. There have been many studies conducted on the modification of MDR in various tumor cell lines, [4][5][6][7][8][9][10] but none is clinically applicable at present. Recently, we have found that photodynamic therapy (PDT) with acridine orange (AO) has a strong cytocidal effect on a chemosensitive mouse osteosarcoma cell line. We conducted the present study to clarify the effect of PDT with AO (AO-PDT) on an MDR mouse osteosarcoma cell line, in comparison with the effect on the chemosensitive cell line. MATERIALS AND METHODS AO-PDT with mouse osteosarcoma cellsThe MDR mouse osteosarcoma cell line (MOS/ADR1) 11) was used in this study. This cell line was established from a radiationinduced mouse osteosarcoma cell line (MOS) 12) by single cell culture after exposure to six-pulsed, stepwise increments of doxorubicin (DOX) concentration ranging from 0.01 to 1 µg/ml. The MOS cells were chemosensitive to most anticancer agents, but the MOS/ADR1 cells showed a classical MDR phenotype with overexpression of P-glycoprotein; they were resistant to DOX, vincristine, vinblastine, etoposide, mitomycin C, and actinomycin D. 11)DOX binding assay demonstrated that more than 90% of the MOS/ADR1 cells were negative for nuclear DOX fluorescence. 11)We cultured 2×10 5 MOS/ADR1 cells and MOS cells in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C under a 5% CO 2 atmosphere, using 6-well plates. At 24 h, in a preconfluent cell growth condition, the medium of the wells was replaced with 0.025, 0.05, 0.1, 1.0, or 2.0 µg/ml AO containing DMEM. After 15-min exposure to AO, 1400 lx blue light selected through an interference filter (466.5 nm) from a 150-W halogen lamp source (Nikon Fibertrans 2; Nikon, Tokyo) was employed to illuminate the cell surface, to excite AO bound to the cells, using a double fiber tube system (PDT with AO: AO-PDT). This blue light has low energy and does not generate heat. In the continuous AO exposure study, after illumination for 10 min, both tumor cell groups were further cultured in an AO-containing medium. In the fl...

Acridine orange (AO) has unique biological actions enabling tumor visualization (fluorovisualization) and a strong cytocidal effect (photodynamic therapy: AO-PDT) under illumination with blue light. Accordingly, in this study, we attempted to develop a new surgical technique for total tumor cell elimination using these photodynamic reactions with AO in a mouse osteosarcoma model. The results showed that local tumor recurrence was significantly inhibited (23%) in the group treated with curettage under fluorovisualization and AO-PDT, compared to that (80%) in the control group treated with curettage alone under ordinary light. Therefore, we concluded that the combination of curettage under fluorovisualization and AO-PDT may be useful for total tumor cell elimination with minimum damage to normal tissue in musculoskeletal sarcomas.

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