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KMID : 0613820090190060770
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Journal of Life Science
2009 Volume.19 No. 6 p.770 ~ p.780
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The Anticancer Effect and Mechanism of Photodynamic Therapy Using 9-Hydroxypheophorbide-a and 660 ㎚ Diode Laser on Human Squamous Carcinoma Cell Line
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Ahn Jin-Chul
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Abstract
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A new photosensitizer, 9-Hydroxypheophorbide-a (9-HpbD-a), was derived from Spirulina platensis. We conducted a series of experiments, in vitro and in vivo, to evaluate the anticancer effect and mechanism of photodynamic therapy using 9-HpbD-a and 660 ㎚ diode lasers on a squamous carcinoma cell line. We studied the cytotoxic effects of pheophytin-a, 9-HpbD-a, 9-HpbD-a red and 660 ㎚ diode lasers in a human head and neck cancer cell line (SNU-1041). Cell growth inhibition was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of 9-HpbD was higher than those of 9-HpbD-a red or pheophytin-a in PDT. We then tested the cytotoxic effects of 9-hydroxypheophorbide-a (9-HpbD-a) in vitro. The cultured SNU-1041 cells were treated with serial concentrations of 9-HpbD-a followed by various energy doses (0, 0.1, 0.5, 3.2 J/㎠) and by various interval times (0, 3, 6, 9, 12 hr) until laser irradiation, then MTT assay was applied to measure the relative inhibitory effects of photodynamic therapy (PDT). Optimal laser irradiation time was 30 minutes and the cytotoxic effects according to incubation time after 9-HpbD-a treatment increased until 6 hours, after which it then showed no increase. To observe the cell death mechanism after PDT, SUN-1041 cells were stained by Hoechst 33342 and propidium iodide after PDT, and observed under transmission electron microscopy (TEM). The principal mechanism of PDT at a low dose of 9-HpbD-a was apoptosis, and at a high dose of 9-HpbD-a it was necrosis. PDT effects were also observed in a xenografted nude mouse model. Group Ⅰ (no 9-HpbD-a, no laser irradiation) and Group Ⅱ (9-HpbD-a injection only) showed no response (4/4, 100%), and Group Ⅲ (laser irradiation only) showed recurrence (1/4, 25%) or no response (3/4, 75%). Group Ⅳ (9-HpbD-a + laser irradiation) showed complete response (10/16, 62.5%), recurrence (4/16, 25%) or no response (2/16, 12.5%). Group Ⅳ showed a significant remission rate compared to other groups (p＜0.05). These results suggest that 9-HpbD-a is a promising photosensitizer for the future and that further studies on biodistribution, toxicity and mechanism of action would be needed to use 9-HpbD-a as a photosensitizer in the clinical setting.
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KEYWORD
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Photodynamic therapy, photosensitizer, 9-Hydroxypheophorbide-a, cell death mechanism, apoptosis
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