Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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The Anticancer Effect and Mechanism of Photodynamic Therapy Using 9-Hydroxypheophorbide-a and 660 nm Diode Laser on Human Squamous Carcinoma Cell Line.

9-hydroxypheophorbide-a와 660 nm 다이오드 레이저를 이용한 광역학치료의 항암효과와 치료기전에 대한 연구

  • Ahn, Jin-Chul (Medical Laser and Device Research Center, Dankook University)
  • 안진철 (단국대학교 의학레이저 의료기기 연구센터)
  • Published : 2009.06.30
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Abstract

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 nm 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 nm 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-I041 cells were treated with serial concentrations of 9-HpbD-a followed by various energy doses (0, 0.1, 0.5, 3.2 J/$cm^{2}$) 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-I041 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.

녹조류인 Spirulina platensis에서 추출하여 만든 새로운 광감작제와 660 nm의 다이오드 레이저를 이용한 광역학치료의 항암효과와 치료기전을 알아보았다. 세포 독성능은 MTT assay를 이용하였고, 세포사멸기전은 propidium iodide과 Hoechst 33342 염색법과 투과전자현미경으로 확인하였다. 또한 암세포가 이종 이식된 누드마우스 모델에서 광역학치료를 시행하여 항암효과를 확인하였다. 3종류의 클로로필 유도체 중 9-hydroxypheophorbide-a (9-HpbD-a)의 세포 독성능이 가장 우수하였고, 9-HpbD-a의 적정 레이저조사 시간은 30분 (3.2 J/$cm^{2}$), 광감작제를 투여하고 레이저조사시간까지의 배양시간은 최소 6시간 이상임을 확인하였다. 광역학치료의 세포사멸기전은 낮은 9-HpbD-a 농도에서 세포고사가 주된 세포사멸기전이었고, 높은 농도의 9-HpbD-a에서는 세포괴사에 의한 세포사멸이 주된 기전임을 확인하였다. 투과전자현미경 하에서도 같은 양상을 관찰하였다. 그리고 암세포가 이종 이식된 누드마우스 모델에서의 광역학치료는 제1군 정상대조군과 제2군 9-HpbD-a만을 투여한 종양조직모두 지속적인 종양의 성장(100% )을 보였고, 제3군인 레이저만을 종양조직에 조사한 실험군에서는 3 마리는 치료가 되지 않았고(75.0%), 1 마리는 재발(25.0%) 하였다. 제4군 광역학치료군에서 총16 마리의 종양에서 10 마리는 완치(62.5%), 4 마리는 재발(25.0%), 2 마리는 치유되지 않았음(12.5% )을 확인하였다. 9-HpbD-a와 660 nm 다이오드 레이저를 이용한 광역학치료는 유의한 항암효과를 나타내었고 9-HpbD-a를 이용한 광역학치료는 새로운 치료방법으로서 향후 암치료의 유용한 치료방법으로 기대된다.

Keywords

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