Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts Ho Jeong Kwon, Minoru Yoshida, Keiichi Abe, Sueharu Horinouchi & Teruhiko Beppu To cite this article: Ho Jeong Kwon, Minoru Yoshida, Keiichi Abe, Sueharu Horinouchi & Teruhiko Beppu (1992) Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts, Bioscience, Biotechnology, and Biochemistry, 56:3, 538-539, DOI: 10.1271/bbb.56.538 To link to this article: http://dx.doi.org/10.1271/bbb.56.538
Published online: 12 Jun 2014.
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Citing articles: 30 View citing articles
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Date: 04 June 2016, At: 13:10
Biosci. Biotech. Biochem., 56 (3), 538-539, 1992
Short Communication
Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts Ho Jeong KWON, Minoru YOSHIDA, Keiichi ABE, Sueharu HORINOUCHI, and Teruhiko BEPPU
Downloaded by [188.68.0.209] at 13:10 04 June 2016
Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University, Bunkyo-ku, Tokyo 113, Japan. Received September 2, 1991
Recent extensive studies on viral and cellular oncogenes have shown that many kinds of protein-tyrosine kinases are important in fundamental cellular regulation such as growth factor-mediated signal transduction, cell division control, and cell differentiation.1. 2 ) The malfunctioning of the protein-tyrosine kinases has also been found to be one of the causes of cell transformation and carcinogenesis. For example, in Rous sarcoma virus (RSV)-infected rat fibroblast cells, morphological changes are induced by a single transforming gene, src, whose product, p60src , has protein kinase activity specific to tyrosine residue. 3,4) To discover new types of antitumor agents, we conducted a screening program for microbial agents that convert the transformed morphology of RSV-infected rat fibroblast cells, SR-3Yl-2 (referred to as SR), to the normal morphology. As a result of screening, we found a potent activity in the culture broth of a fungus, strain KF9. The active agent was identified as radicicol (synonymous with monorden), which had been reported as a macrocyc1ic fungal antibiotic. 5,6) Here, we report the isolation and identification of radicicol as an agent showing a novel biological activity on the src-transformed cell line. Strain KF9 was isolated from a soil sample from Tottori-ken, Japan. It was cultivated in a 5-liter flask containing 1 liter of 1% glucose, 0.5% Polypepton, 0.3% malt extract (Difco), and 0.3% yeast extract (Difco), pH 6.0, at 26°C for 7 days on a rotary shaker at 115 rpm. The active compound was extracted with acetone from'the wet cells (240 g) and was chromatographed on a silica gel column chromatography (Merck, Kieselgel 60, 3 x 15 em) eluted with CHCl 3-methanol (98: 2). The active fractions were collected and further purified by successive HPLC; first, with a column of Senshu Pak Aquasil SS (8cP x 250 mm)
Fig. 2.
using a solvent system composed of CHCI 3-HCOOH (100: 0.5), and second, with a reverse phase column of Senshu Pak SSC-ODS-H-3251 (8cP x 250mm) using methanol-water (7 : 3). The final active fraction was evaporated to dryness to give 32 mg of pure colorless needles called KF9-A substance. The molecular formula of KF9-A was ClsH17CI06 by EI and HRFAB-MS data (MW 364.782). The lH NMR spectrum indicated the presence of two phenolic hydroxy protons (b H = 6.7, bH = 11.2), methine protons of an epoxide (b H =2.96, lH, ddd, J=2.8, 3.2, 8.5Hz; bH =3.l8, lH, s), vinylic protons (b H = 5.84, lH, dd, J = 2, 5, 11 Hz; bH = 6.08, lH, d, J= 16Hz; bH =6.l9, lH, d, J=8, 11 Hz; bH =7.47, lH, dd, J= 11, 16Hz) and methylene protons (b H =2.07, lH, m, J= 15Hz; bH =2.43, lH, ddd, J= 15Hz). The IR spectrum (KBr disc) had a broad band at 3500 em - 1 ascribable to a phenolic hydroxyl group. UVmax were 265 and 315 nm in methanol. All these data indicated that this compound is identical to radicicol. This was confirmed by direct comparison of the 13C·NMR spectral data ofKF9-A with that of authentic radicicol (data not shown).
HO
Fig. 1. Chemical Structure of KF9-A (Radicicol).
Effects of Radicicol on the Morphology of SR Cells.
Shown are a control culture of SR cells (A), a culture treated with 0.5 jlg/ml of radicicol for 12 hr (B), and another control culture of 3YI rat fibroblasts which is the normal counterpart of SR cells (C),
NII-Electronic Library Service
Reversal of src- Transformed Phenotypes by Radicicol
A 1
94
B 2 3 4
123
~
67 43~
Downloaded by [188.68.0.209] at 13:10 04 June 2016
30
~
Fig. 3. Effects of Radicicol on Intracellular Phosphorylation of p60src and Its Synthesis. (A) The p60src in the SR cells treated with radicicol for 12 hr was immunoprecipitated with monoclonal src antibody and used for 10% SDS-polyacrylamide gel electrophoresis. Tyrosine-phosphorylated form of the protein was detected by Western blotting analysis using anti-phosphotyrosine antibody. Lane I, control without radicicol treatment; lane 2, O.llLg/ml; lane 3, 0.5ILg/ml; lane 4, I.0llg/ml. (B) SR cells were pulse-labeled with CSS]methionine (0.3 mCi/ml) for 3 hr and the cell lysate was mixed with the monoclonal src antibody to form an immune complex with p60src . The precipitates containing p60src were analyzed by 10% SDS-polyacrylamide gel electrophoresis and autoradiography. Lane I, control without radicicol treatment; lane 2, 0.5 ILg/ml; lane 3, 1.0 ILg/ml.
SR cells supplied by the JCRB Cell Bank were cultivated in Eagle's minimal essential medium (MEM) supplemented with 12% fetal calf serum at 3rC in a humidified atmosphere of 5% CO 2 , The transformed morphology of SR cells is recognized by their small size with a spindle shape and a loss of contact inhibition, as shown in Fig. 2A. When a small portion of culture broth of strain KF9 was added to SR cells, their morphology became indistinguishable from that of the normal one (Figs. 2B and C). Purified radicicol effectively induced the same morphological reversion at the concentrations of 0.1-1 Jlg/ml. To investigate the mode of action of radicicol, we measured the intracellular level of autophosphorylation at the tyrosine residues in p60src by Western blotting analysis 7) using
539
monoclonal src antibody (Oncogene Science, Inc.) and anti-phosphotyrosine antibody (PY20, ICN Immunobiological). Tyrosine-phosphorylated proteins were detected using streptavidin biotinylated peroxidase complex and an ECL Western blotting detection system (Amersham). As shown in Fig. 3A, radicicol reduced the amount of the phosphorylated form of p60 src in a dose-dependent manner. On the other hand, a pulse-labeling experiment of the radicicol-treated cultures with [ 35 S]methionine showed that synthesis of p60 src was not inhibited significantly by the antibiotic treatment (Fig. 3B). Thus, it is most likely that the reversal of the transformed phenotype of SR cells caused by radicicol is due to the inhibition of p60src in vivo. A variety of microbial metabolites, such as herbimycin,9) erbstatin,lO) and genistein,ll) with no structural similarity, have been reported as inhibitors of tyrosine kinases in vitro. Here we add another agent with a different structure, radicicol, which shows a potent activity in vivo. We assume that the agent may be useful in elucidating the role of the src oncogene in the complicated mechanisms of signal transduction cascades and possibly in cancer chemotherapy. Acknowledgments. We are grateful to Dr. H. Naoki, Suntory Institute for Bioorganic Research, for the spectral analysis, and to Dr. S. Nakajima, Hoshi College of Pharmacy, for his generous gift of authentic radicicol. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan.
References I) 2) 3) 4) 5) 6) 7) 8) 9) 10)
II)
T. Hunter and J. A. Cooper, Annu. Rev. Biochem., 54, 897-930 (1985). Y. Yarden and A. Ullrich, Annu. Rev. Biochem., 57, 433-478 (1988). J. M. Bishop, Annu. Rev. Biochem., 52,301-354 (1983). M. S. Collett and R. L. Erikson, Proc. Natl. Acad. Sci. U.S.A., 75, 2021-2024 (1978). R. N. Mirrington, E. Ritchie, C. W. Shoppee, and W. C. Taylor, Tetrahedron Lett., 7, 365-370 (1964). F. McCapra, A. I. Scott, P. Delmotte, and J. Delmotte-Plaquee, Tetrahedron Lett., 15, 869-875 (1964). J. A. Cooper, B. M. Sefton, and T. Hunter, EMBO J., 4,1471-1477 (1983). A. O. Moria, G. Draetta, D. Beach, and J. Y. 1. Wang, Cell, 58, 193-203 (1989). Y. Uehara, M. Hori, T. Takeuchi, and H. Umezawa, Mol. Cell. BioI., 6,2198-2206 (1986). H. Umezawa, M. Imoto, T. Sawa, K. Isshiki, N. Matsuda, T. Uchida, H. Iinuma, M. Hamada, and T. Takeuchi, J. Antibiotics, 39, 170-173 (1986). H. Ogawara, T. Akiyama, J. Ishida, S. Watanabe, and K. Suzuki, J. Antibiotics, 39, 606-608 (1986).
NII-Electronic Library Service
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts Ho Jeong Kwon, Minoru Yoshida, Keiichi Abe, Sueharu Horinouchi & Teruhiko Beppu To cite this article: Ho Jeong Kwon, Minoru Yoshida, Keiichi Abe, Sueharu Horinouchi & Teruhiko Beppu (1992) Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts, Bioscience, Biotechnology, and Biochemistry, 56:3, 538-539, DOI: 10.1271/bbb.56.538 To link to this article: http://dx.doi.org/10.1271/bbb.56.538
Published online: 12 Jun 2014.
Submit your article to this journal
Article views: 15
View related articles
Citing articles: 30 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [188.68.0.209]
Date: 04 June 2016, At: 13:10
Biosci. Biotech. Biochem., 56 (3), 538-539, 1992
Short Communication
Radicicol, an Agent Inducing the Reversal of Transformed Phenotypes of src-Transformed Fibroblasts Ho Jeong KWON, Minoru YOSHIDA, Keiichi ABE, Sueharu HORINOUCHI, and Teruhiko BEPPU
Downloaded by [188.68.0.209] at 13:10 04 June 2016
Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University, Bunkyo-ku, Tokyo 113, Japan. Received September 2, 1991
Recent extensive studies on viral and cellular oncogenes have shown that many kinds of protein-tyrosine kinases are important in fundamental cellular regulation such as growth factor-mediated signal transduction, cell division control, and cell differentiation.1. 2 ) The malfunctioning of the protein-tyrosine kinases has also been found to be one of the causes of cell transformation and carcinogenesis. For example, in Rous sarcoma virus (RSV)-infected rat fibroblast cells, morphological changes are induced by a single transforming gene, src, whose product, p60src , has protein kinase activity specific to tyrosine residue. 3,4) To discover new types of antitumor agents, we conducted a screening program for microbial agents that convert the transformed morphology of RSV-infected rat fibroblast cells, SR-3Yl-2 (referred to as SR), to the normal morphology. As a result of screening, we found a potent activity in the culture broth of a fungus, strain KF9. The active agent was identified as radicicol (synonymous with monorden), which had been reported as a macrocyc1ic fungal antibiotic. 5,6) Here, we report the isolation and identification of radicicol as an agent showing a novel biological activity on the src-transformed cell line. Strain KF9 was isolated from a soil sample from Tottori-ken, Japan. It was cultivated in a 5-liter flask containing 1 liter of 1% glucose, 0.5% Polypepton, 0.3% malt extract (Difco), and 0.3% yeast extract (Difco), pH 6.0, at 26°C for 7 days on a rotary shaker at 115 rpm. The active compound was extracted with acetone from'the wet cells (240 g) and was chromatographed on a silica gel column chromatography (Merck, Kieselgel 60, 3 x 15 em) eluted with CHCl 3-methanol (98: 2). The active fractions were collected and further purified by successive HPLC; first, with a column of Senshu Pak Aquasil SS (8cP x 250 mm)
Fig. 2.
using a solvent system composed of CHCI 3-HCOOH (100: 0.5), and second, with a reverse phase column of Senshu Pak SSC-ODS-H-3251 (8cP x 250mm) using methanol-water (7 : 3). The final active fraction was evaporated to dryness to give 32 mg of pure colorless needles called KF9-A substance. The molecular formula of KF9-A was ClsH17CI06 by EI and HRFAB-MS data (MW 364.782). The lH NMR spectrum indicated the presence of two phenolic hydroxy protons (b H = 6.7, bH = 11.2), methine protons of an epoxide (b H =2.96, lH, ddd, J=2.8, 3.2, 8.5Hz; bH =3.l8, lH, s), vinylic protons (b H = 5.84, lH, dd, J = 2, 5, 11 Hz; bH = 6.08, lH, d, J= 16Hz; bH =6.l9, lH, d, J=8, 11 Hz; bH =7.47, lH, dd, J= 11, 16Hz) and methylene protons (b H =2.07, lH, m, J= 15Hz; bH =2.43, lH, ddd, J= 15Hz). The IR spectrum (KBr disc) had a broad band at 3500 em - 1 ascribable to a phenolic hydroxyl group. UVmax were 265 and 315 nm in methanol. All these data indicated that this compound is identical to radicicol. This was confirmed by direct comparison of the 13C·NMR spectral data ofKF9-A with that of authentic radicicol (data not shown).
HO
Fig. 1. Chemical Structure of KF9-A (Radicicol).
Effects of Radicicol on the Morphology of SR Cells.
Shown are a control culture of SR cells (A), a culture treated with 0.5 jlg/ml of radicicol for 12 hr (B), and another control culture of 3YI rat fibroblasts which is the normal counterpart of SR cells (C),
NII-Electronic Library Service
Reversal of src- Transformed Phenotypes by Radicicol
A 1
94
B 2 3 4
123
~
67 43~
Downloaded by [188.68.0.209] at 13:10 04 June 2016
30
~
Fig. 3. Effects of Radicicol on Intracellular Phosphorylation of p60src and Its Synthesis. (A) The p60src in the SR cells treated with radicicol for 12 hr was immunoprecipitated with monoclonal src antibody and used for 10% SDS-polyacrylamide gel electrophoresis. Tyrosine-phosphorylated form of the protein was detected by Western blotting analysis using anti-phosphotyrosine antibody. Lane I, control without radicicol treatment; lane 2, O.llLg/ml; lane 3, 0.5ILg/ml; lane 4, I.0llg/ml. (B) SR cells were pulse-labeled with CSS]methionine (0.3 mCi/ml) for 3 hr and the cell lysate was mixed with the monoclonal src antibody to form an immune complex with p60src . The precipitates containing p60src were analyzed by 10% SDS-polyacrylamide gel electrophoresis and autoradiography. Lane I, control without radicicol treatment; lane 2, 0.5 ILg/ml; lane 3, 1.0 ILg/ml.
SR cells supplied by the JCRB Cell Bank were cultivated in Eagle's minimal essential medium (MEM) supplemented with 12% fetal calf serum at 3rC in a humidified atmosphere of 5% CO 2 , The transformed morphology of SR cells is recognized by their small size with a spindle shape and a loss of contact inhibition, as shown in Fig. 2A. When a small portion of culture broth of strain KF9 was added to SR cells, their morphology became indistinguishable from that of the normal one (Figs. 2B and C). Purified radicicol effectively induced the same morphological reversion at the concentrations of 0.1-1 Jlg/ml. To investigate the mode of action of radicicol, we measured the intracellular level of autophosphorylation at the tyrosine residues in p60src by Western blotting analysis 7) using
539
monoclonal src antibody (Oncogene Science, Inc.) and anti-phosphotyrosine antibody (PY20, ICN Immunobiological). Tyrosine-phosphorylated proteins were detected using streptavidin biotinylated peroxidase complex and an ECL Western blotting detection system (Amersham). As shown in Fig. 3A, radicicol reduced the amount of the phosphorylated form of p60 src in a dose-dependent manner. On the other hand, a pulse-labeling experiment of the radicicol-treated cultures with [ 35 S]methionine showed that synthesis of p60 src was not inhibited significantly by the antibiotic treatment (Fig. 3B). Thus, it is most likely that the reversal of the transformed phenotype of SR cells caused by radicicol is due to the inhibition of p60src in vivo. A variety of microbial metabolites, such as herbimycin,9) erbstatin,lO) and genistein,ll) with no structural similarity, have been reported as inhibitors of tyrosine kinases in vitro. Here we add another agent with a different structure, radicicol, which shows a potent activity in vivo. We assume that the agent may be useful in elucidating the role of the src oncogene in the complicated mechanisms of signal transduction cascades and possibly in cancer chemotherapy. Acknowledgments. We are grateful to Dr. H. Naoki, Suntory Institute for Bioorganic Research, for the spectral analysis, and to Dr. S. Nakajima, Hoshi College of Pharmacy, for his generous gift of authentic radicicol. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan.
References I) 2) 3) 4) 5) 6) 7) 8) 9) 10)
II)
T. Hunter and J. A. Cooper, Annu. Rev. Biochem., 54, 897-930 (1985). Y. Yarden and A. Ullrich, Annu. Rev. Biochem., 57, 433-478 (1988). J. M. Bishop, Annu. Rev. Biochem., 52,301-354 (1983). M. S. Collett and R. L. Erikson, Proc. Natl. Acad. Sci. U.S.A., 75, 2021-2024 (1978). R. N. Mirrington, E. Ritchie, C. W. Shoppee, and W. C. Taylor, Tetrahedron Lett., 7, 365-370 (1964). F. McCapra, A. I. Scott, P. Delmotte, and J. Delmotte-Plaquee, Tetrahedron Lett., 15, 869-875 (1964). J. A. Cooper, B. M. Sefton, and T. Hunter, EMBO J., 4,1471-1477 (1983). A. O. Moria, G. Draetta, D. Beach, and J. Y. 1. Wang, Cell, 58, 193-203 (1989). Y. Uehara, M. Hori, T. Takeuchi, and H. Umezawa, Mol. Cell. BioI., 6,2198-2206 (1986). H. Umezawa, M. Imoto, T. Sawa, K. Isshiki, N. Matsuda, T. Uchida, H. Iinuma, M. Hamada, and T. Takeuchi, J. Antibiotics, 39, 170-173 (1986). H. Ogawara, T. Akiyama, J. Ishida, S. Watanabe, and K. Suzuki, J. Antibiotics, 39, 606-608 (1986).
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