Photodynamic Inactivation with Acridine Orange on a Multidrug‐resistant Mouse Osteosarcoma Cell Line

Abstract

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...

Photodynamic therapy of cancer — Challenges of multidrug resistance

“…[15][16][17][18][19] Milla et al showed that PDT-resistant squamous carcinoma cells had a more¯broblastic morphology, higher number of stress¯bers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin but few di®erences in intracellular PpIX content after incubation with ALA methyl derivative. 20 Topical ALA PDT has been successfully used for some resistant cases of cutaneous T-cell lymphoma.…”

In vivo anti-tumor activity of photodynamic therapy with intravenous administration of acridine orange, followed by illumination with high-power flash wave light in a mouse osteosarcoma model

“…Consequently, this in vivo study was conducted. Previously, we reported that the intraperitoneal administration of AO at 10 mg/ kg followed by blue light excitation inhibited the tumor growth of osteosarcoma developing from a different cell line (MOS) than LM8 in vivo (14,15,18,19). Recently, we also found that the intravenous administration of AO at 1.0 mg/kg is useful for PDD of mouse osteosarcomas in nude mice.…”

“…AO was used at a concentration of 1.0 mg/kg, since previous studies showed that this concentration yields the strongest cytocidal effect and lowest toxicity in mice (10). The illumination time (10 min) and time-point of illumination (2 h after AO injection) were also selected based on the results of previous studies (9,10,14,15).…”

Intraoperative Photodynamic Surgery (iPDS) with Acridine Orange for Musculoskeletal Sarcomas

“…Activated oxygen (singlet oxygen) oxidizes the fatty acids of the lysosomal membranes, resulting in leakage of various lysosomal enzymes, such as proteases, lipases, and nucleases, into the cytosolic space. It has been reported that singlet oxygen scavengers, like L-histidine, inhibited the cytocidal effect of AO-PDT [19,36].…”

“…We also demonstrated that AO exerts selective cytocidal effects against sarcoma cells both in vitro and in vivo after illumination with visible light or irradiation of low-dose X-rays. It is available clinically for photodynamic therapy (PDT) [16][17][18][19][20][21] or radiodynamic therapy (RDT) [22][23].…”