Nostoc commune으로부터 백혈병세포와 간암세포에 대한 apoptosis 유도 광과민성물질 pheophytin a의 분리 및 구조동정

Isolation and Identification of Pheophytin, a Photosensitizer from Nostoc commune that Induces Apoptosis in Leukemia and Cancer Cells

Abstract

본 연구에서 N. commune으로부터 U937과 SK-HEP-1 세포에 대해 apoptosis를 유도하는 광과민성물질을 순수 분리하였다. N. commune을 dichloromethane : Methanol (1:1)로 추출하여 Hexane, EtOAc, MeOH로 분획하였다. 높은 광역학활성을 나타내는 EtOAc 분획물에서 NC-1을 분리하였으며, 분자량 870의 pheophytin a ($C_{55}H_{74}N_4O_5$)인 것으로 구조를 동정하였다. 광역학활성 조사는 NC-1을 $1.15{\mu}M$에서 $23.00{\mu}M$까지 5개의 농도를 처리하여 광을 조사하는 것과 조사하지 않는 조건으로 나누어 평가하였다. U937과 SK-HEP-1 두 세포에서 apoptotic body 형성, cell blebbing 등의 형태적 변화와 세포독성, caspase 활성, 세포핵의 응축, DNA 단편화 등이 광 의존적이며 농도 의존적으로 활성이 증가하였다. 추가적으로 유세포분석을 통한 apoptosis 유도의 정량적 분석 결과에서도 광조건에서 농도가 증가할수록 apoptosis 유도가 증가하는 것을 확인하였다. 이러한 결과를 통해 NC-1의 U937과 SK-HEP-1 세포에 대한 사멸은 광역학적 apoptosis에 의한 것임을 확인하였다. 본 연구 내용을 N. commune으로부터 광감각제 개발의 기초자료로 활용할 수 있을 것이다.

The aim of this study was to separate the photosensitizer that induces apoptosis of U937 and SK-HEP-1 cells from Nostoc commune. Dried N. commune was extracted with $CH_2Cl_2/MeOH$ (1:1) to separate the photosensitizer using various chromatographic techniques. The isolated compound was identified as pheophytin a ($C_{55}H_{74}N_4O_5$) with a molecular weight of 870. Its photodynamic activities were assessed under different irradiation conditions (light and non-light) at the same concentration range of $1.15-23.0{\mu}M$. The apoptosis inducing activity in U937 or SK-HEP-1 cells appeared only in the light. The mechanisms underlying the pheophytin a-mediated photodynamic inhibition of cancer cells were further investigated by examining cell morphology changes, cytotoxicity, caspase-3/7 activity, fluorescence staining, flow cytometry analysis, and DNA fragmentation in these two cell lines. The positive control and the light irradiation group showed typical apoptotic responses, including morphological changes, cytotoxicity, caspase activity, nucleus shrinkage owing to chromatin condensation, DNA laddering, and the presence of apoptotic bodies. Cytotoxicity markedly increased in a dose-dependent manner after a 12 hr exposure. Caspase-3/7 activity was higher in U937 cells than in SK-HEP-1 cells. Apoptosis induction therefore appeared to be both concentration- and light-dependent. In conclusion, pheophytin a, isolated from the blue green alga N. commune, had a photodynamic apoptosis-inducing effect on U937 and SK-HEP-1 cells. The findings reported here can be used as basic data for the development of next-generation photosensitizers from N. commune.

Fig. 1. Schematic diagram of isolation process from N. commune.

Fig. 2. TLC developing pattern and HPLC analysis chromatogram of purified compound (NC-1).

Fig. 3. Structure of purified photosensitizer compound (NC-1). NC-1 was determined to be pheophytin a of C₅₅H₇₄N₄O₅.

Fig. 4. Morphological changes in U937 and SK-HEP-1 cells treated with NC-1 and light.

Fig. 5. Growth inhibition effects of U937 and SK-HEP-1 cells by light treatment.

Fig. 6. Measurement of caspase-3/7 activity in U937 and SK-HEP-1 cells by NC-1 and light treatment.

Fig. 7. Fluorescent staining of U937 and SK-HEP-1 cells.

Fig. 8. U937 cells viability by NC-1 treatment and light irradiation.

Fig. 9. SK-HEP-1 cells viability by NC-1 treatment and light irradiation.

Fig. 10. Detection of DNA fragmentation in U937 and SK-HEP-1 cells by NC-1 and light treatment.

Table 1. HPLC operating conditions