The current study is undertaken to investigate the new tumor cell growth inhibitors derived from Streptomyces. The producing organism was isolated from soil collected at the Wonju area (CHIAK mountain) in Kangwon-Do, Korea. The 800 microorganisms were tested in primary screening. Primary screening tests were performed using antimicrobial activity and tumor cell cytotoxicity in vitro. Cultured broth of microorganism was tested for antimicrobial activity against Staphylococcus aureus and for cytotoxicity against P 388 murin cell line. Finally, we chose the SK-302 which has a excellent antimicrobial activity and tumor cell cytotoxicity. The selected microorganism SK-302 belongs to Actinomycetes Streptomyces sp. based on morphological and cultural properties on various medium.
The SK-302 was cultivated in large scale, lab and 300L pilot-scale fermentor, to prepare the sample. Active metabolites were isolateded from cultured broth by several isolation steps of extractions and column chromatography (silica column). Purification was carried out using the Prep-HPLC with ODS column and 1H-2H polystylene gel column. As a result, about 500 mg of pure SK-302B and 27 mg of pure SK-302C were obtained from cultured broth.
Pure SK-302B and SK-302C were tested in vitro (3 LL, B 16 mouse tumor cell), in vivo (P 388, B 16, S-180 mouse tumor cell), in vitro (SK-MEL-2, HCT-15, SNU-1, KATO human tumor cell) tests, based on chemosensitivity tests (cytotoxicity and colony forming inhibition assay).
Structural study of purified fractions were carried out several spectroscopic methods (2D FT-NMR and APCI, FAB MASS) and amino acid analysis. As a result, SK-302B (mol.wt 1101.3) is a cyclooctapeptide (essential amino acid: Ala, Ser, non-essential amino acid N-Me Cys, N-Me Val) with aromatic (quinoxaline) moiety. The candidate compound for antitumor agent, SK-302B, was finally identified as a known compound, echinomycin A (quinomycin A).
Echinomycin and It's synthetic analogue TANDEM is tested in phase II trial. But the biggest problem of echinomycin is a strong cytotoxicity to the normal cells. In this study, the aim of solving this problem, several synthetic echinomycin analogues (sulfoxide, sulfone and sulfoniun salts) were synthesized. Echinomycin sulfoxide (mol. wt 1117) disulfoxide(mol. wt 1133) echinomycin sulfone (mol. wt 1133) and S-methylated sulfonium perchlorate (mol. wt 1216) were identified by Mass(APCI and ESI), FT-IR and FT-NMR ($^1H$,$^13C$ and 2D NOESY).
Newly modified echinomycin such as S-methylated sulfonium perchlorate, sulfoxide, disulfoxide and sulfone have been prepared and evaluated for in vitro biological activities of cytotoxicity against P 388, B 16 and SNU 16 as well as in vivo antitumor activities against murine leukemia P 388 and melanoma B 16. Of these compounds, S-methylated sulfonium perchlorate inhibits P 388 proliferation in vitro stronger than adriamycin and echinomycin and increase the life span of tumor (P 388 and B 16) bearing mice as high as compared to that of echinomycin at the therapeutic dose.
The present study showed that the modified echinomycin such as S-methylated sulfonium perchlorate could have more excellent antitumor activity with the lower toxicity than that of echinomycin.
원주 치악산에서 채취된 방선균으로부터 강한 항암작용을 보이는 물질을 분리하여 항암력을 측정한 결과 P 388 세포에서 강한 항암작용을 나타내는 물질 SK-302B를 분리할수 있었다. 항암작용을 나타내는 물질의 구조를 규명하기위해 300 L 규모의 배양기에서 SK-302B를 배양한후에 칼럼크로마토그라피를 이용하여 순수한 물질 500 mg을 얻을수 있었다. 구조를 규명하기위해 아미노산분석, 이차원 핵자기공명분석기, 질량분석기를 통하여 SK-302B는 이미 알려진 이치노마이신임을 확인할 수 있었다. 이치노마이신은 항암력은 우수하나 정상세포에 대한 독성이 강한 문제점을 가지고 있다. 이런 문제점을 해결하기 위해 새로운 형태의 이치노마이신 유도체인 술폰, 술폭사이드 및 술포니움염을 합성하였다. 새로 합성된 이치노마이신 유도체는 이치노마이신보다 극성이 강하여 용매에 대한 용해도를 증가 시킬수 있었다. 합성된 이치노마이신 유도체를 P 388, B 16 및 SNU 16 암세포에 대해 항암력시험을 실시하였다. 시험결과 특히 이치노마이신의 술포니움염에서 항암력은 유지가 되면서, 독성이 크게 줄어드는 결과를 얻을수 있었다. 이치노마이신의 술포니움염은 항암작용에 대한 기작도 큰차이를 보여 이에 대한 연구를 진행중에 있다.
