Obstetrics & gynecology science

Korean Society of Obstetrics and Gynecology (대한산부인과학회)
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The anticancer effect and mechanism of photodynamic therapy using a new photosensitizer and diode laser on cervical cancer cell

자궁경부암 세포주에서 새로운 광감작제와 다이오드 레이저를 이용한 광역학치료의 항암효과와 치료기전에 대한 연구

Kim, Jong-Soo;Park, Choong-Hak;Chung, Phil-Sang
김종수;박충학;정필상

  • Published : 20090000
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Abstract

Objective: During the last several years, such as Photofrin(R), Photogem(R), HpD, and 5-aminolevulenic acid (ALA) has been used as photosensitizers for a wide range of malignant tumors as well as non-malignant disease. A new photosensitizer, 9-Hydroxypheophorbide-a (9-HpbD-a) was derived from Spirulina platensis. The aim of this study was to evaluate the anticancer effect and mechanism of photodynamic therapy in vitro using 9-HpbD-a and 670 nm diode laser on a HT-3 cervical cancer cell line. Methods: We studied the cytotoxic effect of 9-HpbD-a and 670 nm diode laser in HT-3 cervical cancer cell line. The cultured HT-3 cells were treated with serial concentrations of 9-HpbD-a followed by various irradiation time (0, 5, 15, 30 min) and by various interval times (0, 3, 6, 9, 12 24 hours) until laser irradiation, then 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay was applied to measure the relative inhibitory effect of PDT. Results: Optimal condition for laser irradiation time was 15 minutes and the cytotoxic effect according to the incubation time after 9-HpbD-a treatment was increased until 6 hours. Under confocal microscopy, to observe intracellular localization of 9-HpbD-a, cells dual-stained with 9-HpbD-a and panel of organelle-specific fluorescence probes (MitoTracker, LysoTracker, ER-Tracker), endoplasmic reticulum (ER) as a major accumulation organelle site in HT-3 cervical cell line for this new photosensitizer. Conclusion: The anticancer effect of PDT using 9-HpbD-a is effective in HT-3 cervical cancer cell line. This agent therefore represents a promising photosensitizing prodrug for the treatment of cervical cancer with PDT in clinical setting for the future.

목적: 광역학치료에서 기존에 사용 중인 광감작제의 단점을 보완하여 새로운 재료에서 높은 파장의 흡수를 보이고 빠른 대사를 보이는 보다 효과적인 암세포를 죽일 수 있는 광감작제의 개발이 이루어지고 있는데, 최근 국내에서 개발된 광감작제로 녹조류인 Spirulina platensis에서 추출하여 만든 광감작제 9-hydroxypheophorbide-a (9-HpbD-a)를 이용하여 자궁경부암 세포주인 HT-3에서 새로 개발된 9-HpbD-a 광감작제의 임상적용을 위하여 국내에서 개발된 670 nm의 다이오드 레이저를 이용한 광역학치료의 항암효과 및 치료기전을 확인하고자 본 연구를 시행하였다. 연구 방법: 광감작제는 금호 생명과학연구소에서 녹조류의 클로로필을 산화반응과 산 처리를 통해 10-hydroxypheophytin-a 유도체로 만든 다음 이를 유기용매를 이용하여 추출한 10-hydroxypheophytin과 10-hydroxypheophytin-a 유도체를 유기합성 후 안정된 구조 및 대량 수율이 가능한 9-hydroxypheophorbide-a (9-HpbD-a)를 사용하였다. 세포주는 인체의 자궁경부 편평세포암주 (squamous cervical cancer cell)인 HT-3을 사용하였다 (ATCC(R)Number: HTB-$32^{TM}$). 광감작제와 레이저조사가 세포에 미치는 독성능을 알아보기 위해 MTT 검색법을 이용하였으며, 9-HpbD-a의 세포 내 축적위치를 확인하기 위하여 세포 내 소기관에 특이적으로 염색되는 형광시약과 9-HpbD-a를 이 중 염색하여 confocal microscope를 이용하여 확인하였다. 결과: HT-3 자궁경부암 세포주에서 클로로필 유도체인 9-HpbD-a의 세포독성능을 검사한 결과 생체 외 (in vitro) 실험에서 9-HpbD-a의 농도와 레이저의 조사량 그리고 9-HpbD-a의 투여 후 레이저조사까지의 시간 간격이 증가할수록 세포생존율이 통계적으로 의미 있게 감소하였다. 9-HpbD-a의 적정 레이저조사 시간은 15분 (1.98 J/cm^{2}), 광감작제를 투여하고 레이저조사 시간까지의 배양시간은 최소 6시간 이상임을 확인하였다. 광학현미경상으로 광감작제만을 투여한 군과 레이저조사만을 한 군에서는 HT-3 세포의 형태학적 변화는 나타나지 않았으며, 광역학치료 군에서는 세포가 괴사되는 형태학적인 변화를 보였다. Confocal microscopy를 이용하여 세포 내 9-HpbD-a의 축적 소기관을 확인 하였는데, 9-HpbD-a가 세포 내 소포체에 가장 많이 축적되고 미토콘드리아와 라이소좀에서는 약간의 축적됨을 확인하였다. 결론: 본 연구에서 이용한 9-HpbD-a가 기존에 사용하고 있는 1세대 광감작제를 대신하여 효과적인 광감작제로 사용되기 위해서는 종양조직에만 많은 양의 광감작제가 선택적으로 축적되게 하여 광역학치료의 효과를 높여야 하며, 9-HpbD-a의 독성능 시험을 비롯하여 현재 사용 중인 항암제와의 병합요법에 대한 연구 및 9-HpbD-a를 이용한 광역학진단 (photodynamic diagnosis) 연구를 토대로 새로운 광감작제와 함께 인체조직의 침투력이 좋은 우수한 레이저의 개발도 함께 이루어져야 한다.

Keywords

References

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