Mechanism of photosensitization by microsphere-bound chlorin e6 in human bladder carcinoma cells.
Photodynamic therapy is an experimental method of cancer treatment in which a photosensitizer is administered and subsequently the tumor is irradiated with light. Due to problems of prolonged skin phototoxicity with hematoporphyrin derivative, new photosensitizers and methods of localization are being sought. The goal of this study was to compare the photosensitizer chlorin e6 (Ce6) free and bound to 1-micron-diameter microspheres (MS) for phototoxicity, uptake and efflux characteristics, phagocytosis rates in malignant and benign cells, and effects of NaN3, D2O, and buthionine sulfoximine on phototoxic efficacy. Incubation of MGH-U1 human bladder carcinoma cells with Ce6-MS (0.43 microM Ce6-equivalent; 18 h) and subsequent irradiation using an argon laser-pumped dye laser at a radiant exposure of 20 J/cm2 caused 100% cell death 24 h after irradiation. In contrast, MGH-U1 cells incubated with free Ce6 (0.43 microM; 18 h) remained 100% viable 24 h after irradiation at a radiant exposure of up to 50 J/cm2. The presence of D2O during irradiation increased the phototoxicity to MGH-U1 cells, whereas the presence of NaN3 decreased it; these data support an important role for 1O2. Irradiation of MGH-U1 cells in the presence of the glutathione depleter buthionine sulfoximine also increased the phototoxicity, demonstrating a role for intracellular glutathione and possibly free radical intermediates. The cellular uptake of Ce6 was approximately 50 times lower than that of Ce6-MS at equivalent incubation concentrations. Efflux experiments showed that the phototoxicity of Ce6-MS was reduced by 40% for a 5-h washout time as compared to no washout time. In contrast, for free Ce6, the decrease was 95.3% under identical conditions. Because the total intracellular concentration of Ce6-MS after an efflux time of 5 h was only slightly changed, the decreased phototoxicity is attributed to an altered intracellular localization. Confocal laser scanning fluorescence microscopy data appear consistent with this hypothesis although they are not conclusive. The observed patterns were different at 0 and 5 h. Comparison of the phagocytosis rates of Ce6-MS by carcinoma and benign cells showed that on average 20 MS/cell were phagocytosed by MGH-U1 compared with 2.5 and 8.3 in the benign human fibroblasts and keratinocytes, respectively. After incubation with Ce6-MS (0.1 microM; 18 h) and irradiation at 10 J/cm2 the surviving fraction of MGH-U1 cells was 76.3 +/- 0.95% (mean +/- SE) and 40 +/- 3.49% for fibroblasts. In contrast, for keratinocytes the surviving fraction was 93.5 +/- 0.83%.(ABSTRACT TRUNCATED AT 400 WORDS)
Bachor
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Wellman Laboratories of Photomedicine, Department of Urology, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Scholz
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Shea
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CR
Hasan
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D019328:Buthionine Sulfoximine; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002735:Chlorophyllides; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007700:Kinetics; D008027:Light; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008863:Microspheres; D011166:Porphyrins / Q000494:pharmacology*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D001749:Urinary Bladder Neoplasms
D002735:Chlorophyllides; D011166:Porphyrins; D011838:Radiation-Sensitizing Agents; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; C062985:phytochlorin