The Streptomyces sp. MCH79 was selected from the cytotoxicity assay against tumor cells to search for novel antitumor compound from microbial metabolites. This strain was identified as Streptomyces ostreogriseus on the basis of its cultural, morphological and physiological properties. The active compounds, MCH-14 (40.1 mg), MCH-15 (26.9 mg), MCH-16 (20.1 mg), MCH-17 (91.7 mg), MCH-18 (21.6 mg), MCH-22 (135.2 mg), MCH-31 (52.2 mg), MCH-23 (25.4 mg), MCH-32 (19.6 mg), MCH-Vi (9600 mg) and MCH-Vj (143.4 mg), were recovered from the mycelium of fermentation broth (120ℓ)with acetone extraction followed by purification on a silica gel column chromatography and reverse-phase HPLC chromatography. The structure of MCH compounds were determined by spectroscopic methods of UV, FT-IR, $^1H$-NMR, $^{13}C$-NMR, DEPT, $^1H-^1H$ COSY, $^{13}C-^1H$ COSY, HMBC and FAB-MS as well as physicochemical data such as melting point and optical rotation. Structural studies revealed that there were seven novel oligomycin analogues, 41-demethyl-29 -hydroxy-homooligomycin A, 41-demethyl-oligomycin E, 41-demethyl-44-hydroxy-homooligomycin A, 41-demethyl-homooligomycin E, 41-demethyl-homooligomycin B, homooligomycin E, 41-demethyl-homooligomycin A and four known oligomycins, 44-homooligomycin B, 44-homooligomycin A, oligomycin A, 44-homooligomycin C. The MCH compounds showed selective growth inhibition against cell lines such as RHEK-1 (human epidermal keratinocyte cell line), RHEK-1/pSV2 ras (cell line transformed with $pSV_2$ras), NIH-3T3 (NIH Swiss mouse embryo fibrolast), F25 (oncogene ras transformed NIH-3T3) and human tumor cell lines, SNU-1 (human stomach cancer) and SNU-354 (human liver cancer), with the $IC_{50}$ values range of 0.073-19.0, 0.025-6.50, 1.17-159.75, 0.22-0.75, 0.17-0.63, 0.19-1.05㎍/ml, respectively. The MCH compounds showed stronger cytotoxic effect on RHEK-1/pSVras and F25 than on their parent cells, RHEK-1 and NIH-3T3, indicating that the compounds conferred differential cytotoxicity between ras-transformed and its parent cells. The MCH compounds also showed potent immunosuppresive activities on T-dependent antibody response against sheep red blood cell, polyclonal in B cell activation by lipopolysaccharide (LPS) and T cell activation in a mixed lymphocyte reaction. By comparison with vehicle control (0.01% DMSO), the MCH compounds exhibited a strong immunosuppression on immune responses at concentration range from 1.0 to 0.01 ㎍/ml. The proliferation of splenic lymphocytes was induced by B cell mitogen such as LPS, T cell mitogens such as concanavalin A (Con A) and phytohemagglutinin (PHA), and T/B cell common mitogen such as pokeweed mitogen (PWM). By the treatment of the MCH compounds, the induced incorporation of [$^3H$]-thymidine into DNA of lymphocytes was inhibited and the suppression on B cell proliferation was more extensive than T cells. In conclusion, these data indicated that the MCH compounds had a potent growth inhibition against various human tumor cell lines and a potent suppressive activity on the functions of splenic lymphocytes.
The anthracycline antibiotics such as daunorubicin, doxorubicin and aclacinomycin are clinically and commercially important anticancer agents. Their cardiotoxic nature limits their dose and long-term use. This major drawback has led to an intensive search for new members of anthracycline either by a screening of blocked mutants or by a hybrid biosynthetic approach. In the course of study on the biosynthesis of doxorubicin, aklavinone 11-hydroxylase gene (dnrF) from doxorubicin-producing Streptomyces peucetius subsp. caesius ATCC 27952 was introduced into Streptomyces galilaeus ATCC 31133, an aklavinone glycoside producer by the methods of protoplast transformation. Many reddish metabolites were purified from the culture of the transformant by preparative silica TLC and ECONOSIL SILICA HPLC column chromatography and their structures were identified as 11-hydroxyaclacinomycin A (56 mg), 11-hydroxyaclacinomycin B (35 mg) and 11-hydroxyaclacinomycin T (7 mg). In addition, a new a nthracyline 2'''-amino-11-hydroxyaclacinomycin Y (7-0-[0-α-L-rednosyl- (1→4)-2-deoxy-0-α-L-fucosyl-(1→)-α-L-rhodosam-inyl]-ε-rhodomycinone) was isolated and named it 11-hydroxy aclacinomycin X (43 mg). These results indicated not only that dnrF was stably expressed in S. galilaeus ATCC 31133 to introduce hydroxyl group at C-11 of aglycon but also that the host strain, S. galilaeus ATCC 31133 produced 2"-aminoaclacinomycin Y. Accordingly this compound was also purified from the wild type strain and structure was determined to be a new anthracycline compound as 7-(O-rhodosaminyl-deoxyfucosyl-rednosyl)-aklavinone and was named aclacinomycin X. The in vitro cytotoxicity of 11-hydroxyaclacinomycin X and aclacinomycin X for various types of human tumor cell lines were tested by the Developmental Therapeutic Program of National Cancer Institute, U.S.A. in order to evaluate the drug's selectivity for particular tumor types. 11-Hydroxyaclacinomycin X showed very active and selective cytotoxicities against certain cell lines of leukemia (HL-60), non-small cell lung cancer (NCI-H226), colon cancer (COLO 205), CNS cancer (SF-539), melanoma (MALME-3M) and renal cancer (CAKI-1), with the average total growth inhibition ($log_{10} TGI)$ of <-8.00, <-8.00, -7.89, -7.65, <-8.00, <-8.00 M, respectively. Aclacinomycin X also showed potent cytotoxicities of leukemia (MOLT-4), NCI-H226, colon cancer (HCC-2998), melanoma (SNB-75) and CAKI-1, with the $log_{10}TGI$ of <-8.00, -7.05, -7.53, -6.26, -7.44, -7.30 M, respectively. The cytotoxicity results indicate that the hybrid antibiotic, 11-hydroxyaclacinomycin X is more toxic than aclacinomycin X by 1 to several orders of magnitude. These results suggest that a hydroxyl group at C-11 of aglycon may play an important role in biological activities. These remarkably potent cytotoxicities and the high sensitivity of certain human cancer cells suggest that this compound should be further evaluated for future application in cancer chemotherapy.
항종양 항생물질을 탐색하기 위하여 우리나라 각지의 토양으로 부터 방선균을 약 1500종 이상을 분리하였다. 그중 우리나라 사람으로 부터 분리한 위암세포주 SNU-1과 간암세포주 SNU-354에 세포독성을 보이고 동시에 사람 표피세포 RHEK-1과 RHEK-1에 H-ras oncogene으로 transfomed된 세포준 RHEK-1/pSV2ras에 대하여 다른 세포독성을 나타내는 활성물질을 지닌 반성균 8 균주를 분리하였다. 이중 Streptomyces sp. MCH79 선택하여 이로부터 활성물질 11 종류를 분리정제 하였다. 이 균주를 배양학적, 형태학적, 생리학적 성질을 기초로 하여 Streptomyes costreogriseus로 동정 하였다.
이 항종양 항생물질을 Streptomyces sp. MCH79 발효액 (120 liters)으로 부터 acetone 용매 추출, $SiO_2$ column chromatography와 HPLC column chromatography를 이용하요 분리정제 하였으며, 백색 분말 상태의 순수한 화합물 MCH-14 (40.1 mg), (135.2 mg), MCH-23 (25.4 mg), MCH-31 (52.2 mg), MCH-32 (19.6 mg), MCH-Vi (9600 mg),MCH-Vj (143.4 mg)를 얻었다. MCH 화합물의 구조는 UV, FT-IR, $^H$-NMR,$^13C-NMR, DEPT, $^1H-^1H$COST, $^13C-^1H$ COSY, HMBC, FAB-MS 같은 기기분석 방법으로부터 새로운 화합물 7종류, MCH-14 (41-demethyl-29 hydroxy-homooligomycin A, $C_45H_74O)12$), MCH-15 (41-demethyl-oligomycin E, $C_44H_70O_13$), MCH-16 (41-demethyl-44-hydroxy-homooligomycin A, $C_45H_74O_12$), MCH-17 (41-demethyl-homooligomycin E, $C_45H_72_O_13$), MCH-18 (41-demethyl-homooligomycin B, $C_45H_72_O_12$), MCH-22 (homooligomycin E, $C_46H_74_O-13$), MCH-31 (41-demethyl-homooligomycin A, $C_45H_74O_11$)와 기존 화합물 4종류, MCH-23 (44-homooligomycin B, $C_46H_74O_12$), MCH-32 (oligomycin A, $C_45H_74O_11$), MCH-Vi (44-homooligomycin A, $C_46H_74O_11$), MCH-Vj (homooligomycin C, $C_46H_76O_10$)의 구조를 결정하였다. $^13C$-NMR data로 부터 MCH-14, MCH-31, MCH-32, MCH-Vi, MCH-Vj는 세개의 이중 결합과 세개의 carbonyl group을 가지며, 나머지는 네개의 carbonyl group을 지닌다. $^1H-^1H$ COSY, $^13C-^1H$ COSY, HMBC 기기 분석으로부터 MCH-14, MCH-16, MCH-17, MCH-18, MCH-31은 conjugated diene과 26 membered $\alpha, beta$-unsaturated lactone을 지닌 bicyclic spiroketal moiety와 하나의 ethyl group을 가지며 나머지는 두개의 ethyl group을 지니고 있다. $^1-H-^1H$ COSY와 $ ^13C-^1H$ COSY data에서 C-20과 C-26주위의 proton pattern이 oligomycin A와는 상당이 다르다. HMBC 기기 분석 결과, 41-demethyl 구조를 지니는 화합물은 C-41과 H-19, H-20, H-21는 correlations을 보이므로 C-20에 ethyl 잔기 대신에 methyl 잔기로 치환된 구조를 지니고 있다. 또한 HMBC 기기 분석 결과, 44-homo 구조를 지니는 화합물은 C-44'와 H-66, H-44는 correlation을 보이므로 C-26에 ethyl group가지고 있다.
MCH 화합물은 RHEK-1, RHEK-1/pSV2 ras, NIH-3T3 (NIH Swiss mouse embryo fibroblast), F25 (Oncogene ras transformed NIH-3T3), SNU-1 그리고 SNU-254 세포주에 fibroblast), F25(oncogene ras transformed NIH-3T3), SNU-1그리고 SNU-354 세포주에 대하여 농도 의존적으로 세포독성을 나타내었다. 각 세포주에 대한 저해 농도치 ($IC_50$)범위는 0.073-19.0, 0.025-6.50, 1.17-159.75, 0.22-0.75, 0.17-0.63, 0.19-1.05 ㎍/ml이었다. SNU-1과 SNU-354 대한 세포 독성은 adriamycin 과는 비슷하고 taxol 보다도 나은 효과를 보였다. RHEK-1 과 NIH-3T3보다는 ras oncogene 이 transformed 된 RHEK-1/pSVras 과 F25 세포주에 대하여 강력한 세포독성을 보였다. MCH 화합물들은 면양 적혈구 세포에 대한 T-dependent 항체 생성,lipopolysaccharide (LPS)의한 B세포 활성 및 혼합 림파구 반응에 대한 T세포 활성이 1.0에서 0.01 ㎍/ml 범위에서 강력한 면역억제 활성을 보였다. 또한 LPS, concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM)와 같은 mitogen에 의한 비장 림파구의 분화 유발을 1.0에서 0.01 ㎍/ml 농도 범위에서 강력하게 억제하였다. 특히 T세포 보다는 B 세포 분화를 더욱 저해 하였다. 결론적으로 MCH 화합물들은 항종양 세포 억제 작용과 면역억제 효과를 보였다.
Anthracycline 항생제인 daunorubivin, doxorubicin 그리고 aclacinomycin은 임상적으로 상용되는 매우 중요한 항암 약제이다. 이들 약물은 심장 독성 및 장기간 투여에 상당한 문제점이 있다. 이러한 문제점은 blocked mutants 와 hybrid 생합성 기술을 이용하여 새로운 약물을 만들어 극복할 수가 있다. 본 실험실에서는 doxorubicin 생합성 연구 과정중에 doxorubicin 생성균주 Streptomyces peucetius subsp. caesius ATCC 27952로 부터 aklavinone 11-hydroxylase gene (dnrF)를 포함하는 plasmid pMC213 을 S. galilaeus ATCC 31133에 transformation 하였다. Transformant의 배양액으로부터 많은 적색의 대사산물을 분리하고 구조를 결정하였다. 기기 분석을 통하여 11-hydroxyaclacinomycin A (56mg), 11-jydroxyaclacinomycin B(35mg), 11-hydroxyaclacinomycin T (7mg)을 결정하였으며, 새로운 anthracyline, 3"-amino-11-hydroxyaclacinomycin Y (7-0-[0-α-L-rednosyl-(1→4)-2-deoxy-O-α-L-fucosyl-(1→4)-α-L-rhodosam-inyl]-ε-rhodomycinone)을 분리하여 11-hydroxyaclacinomycin X (43mg)로 명명 하였다. 이러한 결과로 부터 S.galilaeus ATCC 31133 에 dnrF 유전자를 유입시키면 aglycon (aklavinone) C-11 위치애 hydoxyl group이 안정적으로 발현되는 현상을 발견하였으며 또한 본 균주인 S. falilaeus ATCC 31133에 2"-aminoaclacinomycin Y(7-(O-rhodosaminyl-deoxyfucosyl-rednosyl)-aklavinone)이 존재한다는 사실도 발견하여 aclacinomycin X (55mg)를 분리 정제하여 구조를 결정 하였다. 11-aclacinomycin X와 aclacinomycin X 대한 in vitor 세포독성을 미국 National Cancer Institute의 60종의 사람 종양 세포주들에 대하여 세포 독성실험을 실시하였다. 11-Hydroxyaclacinomycin X는 백혈병 (HL-60), 폐암 (NCI-H226), 결장암 (COLO 205), 중추신경계암 (SF-539), 흑생종암 (Malme-3M0, 신장암 (CAKI-1)에 대하여 선택적인 세포독성을 나타내고 각각의 $log_10$TGI (total growth inhinbition)는 <-8.00. <-8.00, -7.89, -7.65, <-8.00, <-8.00 M이었다. Aclacinomycin X는 백혈병 (MOLT-4), NCI-H226, 결장암 (HCC-2998), 흑생종암 (SNB-75), CAKI-1에 대하여 선택적인 세포독성을 나타내고 각각의 $log_10$TGI는 <-8.00, -7.05, -7.53,-6.26, -7.44, -7.30 M 이었다. 이 같은 세포 독성으로부터 hybrid 항생물질인 11-hydroxyaclacinomycin X는 aclacinomycin X보다 대부분의 세포주에 대하여 10배 이상 강력한 항암활성을 나타내었다. 이 같은 결과로 부터 aglycon의 C-11에 hydroxyl group의 유입은 생물학적 활성에 중요한 역할을 하고 있음을 발견하였다.
