Anti-angiogenic Activity and Intracellular Distribution of

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Biol. Pharm. Bull. 34(3) 396—400 (2011)

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Anti-angiogenic Activity and Intracellular Distribution of Epigallocatechin-3-gallate Analogs Suratsawadee PIYAVIRIYAKUL,a,c Kosuke SHIMIZU,a Tomohiro ASAKAWA,b Toshiyuki KAN,b Pongpun SIRIPONG,c and Naoto OKU*,a a

Department of Medical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka; b Department of Synthetic Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526, Japan: and c Research Division, National Cancer Institute Thailand; 268/1 Rama 6, Rajthavee, Bangkok 10400, Thailand. Received October 9, 2010; accepted November 27, 2010; published online December 8, 2010 Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 m M. To elucidate antiangiogenic effect of EGCG, we used a 1 m M concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria. Key words: green tea catechin; ()-epigallocatechin-3-O-gallate; angiogenesis; intracellular distribution; mitochondria

Tumor angiogenesis, a process of new blood vessel formation from pre-existing vessels, is crucial for the growth and development of tumors. It requires multiple interactions among endothelial cells, surrounding pericytes, and smooth muscle cells, extracellular matrix, and angiogenic cytokines/growth factors.1—3) Since effective inhibition of tumor angiogenesis promises to provide crucial suppression of not only tumor growth but also tumor metastasis, the development of agents inhibiting angiogenic processes has become a matter of focus. ()-Epigallocatechin-3-O-gallate (EGCG) is known as a major component of green tea extracts and to exhibit various biological activities including anti-viral, anti-microbial, and anti-oxidative ones.4—8) Especially, the relationship between EGCG and anti-cancer activity is now the most notable, since cancer is the leading cause of human death in various countries. Previous studies demonstrated that EGCG suppresses tumor growth through the inhibition of tumor angiogenesis, and mechanistic studies on this anti-angiogenic effect of EGCG is now ongoing. Tang et al. demonstrated that EGCG inhibits vascular endothelial growth factor (VEGF)-induced Akt activation and VE-cadherin phosphorylation9); and Rodriguez et al., that green tea catechin disrupts VEGF/VEGF receptor (VEGFR) complexes and subsequent signal transduction.10) Also, Tachibana et al. directly identified the target molecule of EGCG as a 67-kDa laminin receptor by using surface plasmon resonance (SPR) assay, and they observed the suppression of tumor angiogenesis through the inhibition of the receptor-mediated signal transduction.11,12) Our previous studies also demonstrated that EGCG suppresses tumor angiogenesis through ∗ To whom correspondence should be addressed.

the inhibition of membrane-type1 matrix metalloproteinase (MT1-MMP).13,14) These findings indicate that EGCG, as a functional component of green tea, will become useful for cancer therapy and chemoprevention. Previous study demonstrated that 5,7-deoxyepigallocatechin gallate (DOEGCG), a synthetic EGCG derivative, possesses more potent anti-influenza infection activity than the original EGCG, suggesting that the phenolic hydroxyl groups on the A-ring in the flavan structure are not involved in the biological activity and that modification of A-ring of the EGCG structure does not abolish but rather enhances the functional ability of EGCG.15) Based on this and other structure–relationship studies,16,17) we synthesized ()-6(5-aminopentyl)-5,7-deoxyepigallocatechin gallate (cisAPDOEGCG) and ()-6-(5-aminopentyl)-5,7-deoxygallocatechin gallate (trans-APDOEGCG) as EGCG derivatives containing linkers and reactive amino groups added to the EGCG structure, as shown in Fig. 1. We compared their antiangiogenic activity with that of EGCG by examining the inhibitory effect on proliferation, migration, invasion, and tube formation by HUVECs. Then, we synthesized fluorescencelabeled EGCG by conjugating the fluorescent reagent TokyoGreen (TG) to ()-cis-APDOEGCG (EGCG-TG) and examined the intracellular distribution of it in HUVECs by confocal laser scanning microscopy. MATERIALS AND METHODS Materials EGCG was purchased from Wako Pure Chemical Industries (Tokyo, Japan). EGCG derivatives, i.e.,

e-mail: [email protected]

© 2011 Pharmaceutical Society of Japan

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Fig. 1. Structures of EGCG and Its Derivatives

cis- and trans-APDOEGCG, and TokyoGreen (TG) were synthesized as described previously.18—20) TG conjugation to ()-cis-APDOEGCG was also described earlier, and the obtained EGCG-TG was purified by HPLC.18) All other reagents used were of analytical grade. Cell Culture Primary human umbilical vein endothelial cells (HUVECs, Lonza, Walkersville, MD, U.S.A.) were cultured in endothelial cell growth medium-2 (EGM-2, Lonza) containing heat-inactivated 2% fetal bovine serum (FBS), human VEGF, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGFB), ascorbic acid, heparin, hydrocortisone, gentamycin sulfate, and amphotericin-B at 37 °C in the presence of 5% CO2 in a humid atmosphere. Anti-proliferative Assay HUVECs (7.5103 cells/well) were seeded onto a 24-well plate pre-coated with 0.1% gelatin. Twenty-four hours later, EGCG or EGCG derivatives (cis- or trans-APDOEGCG) at final concentrations of 0, 1, 3, 10, 30, and 100 m M were applied to each well; and the cells were then incubated for 48 h. TetraColor One solution (Seikagaku Biobusiness Corporation, Tokyo, Japan) was added to each well; and the cells were further incubated for 4 h. Then, the absorbance at the test wavelength of 450 nm and reference wavelength of 630 nm were measured by using a microplate reader (Infinite M200, Tecan Japan, Kanagawa, Japan). Motility and Invasion Assays Motility and invasion assays were performed by a modification of a previously described method.21) In brief, HUVECs in serum-free endothelial cell basal medium-2 (EBM-2, Lonza) were fluorescently labeled with 3 m M 3-O-acetyl-2-7-bis(carboxyethyl)-4 or 5carboxyfluorecein, diacetoxymethylester (BCECF-AM, Dojindo Laboratories, Kumamoto, Japan) for 30 min at 37 °C and then washed with the same medium lacking the label. The cells (5104 cells in 300 m l of EBM-2) were applied to a FALCON HTS FluoroBlokTM Insert (BD, Franklin, Lakes, NJ, U.S.A.), non-coated for the motility assay or pre-coated with BD Matrigel (125 m g/insert, BD) for the invasion assay. Each culture insert was set into a well containing 700 m l of EBM-2 supplemented with 10% FBS (Japan Bio Serum, Hiroshima, Japan). Then, EGCG, cis- or trans-APDOEGCG at a concentration of 1 m M was added to the insert, and the inserts were incubated for 24 h at 37 °C. After the incubation, the FluoroBlok membrane was picked up and placed on a

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glass slide, and the cells that invaded to the lower side of the membrane were observed under a fluorescence microscope (IX71, Olympus, Tokyo, Japan) equipped with a CCD camera (Penguin 600CL, Pixera, Osaka, Japan). Capillary Tube Formation Assay Matrigel tube formation assay was performed as follows: BD Matrigel (4 mg/ml) was added to each well of a 24-well plate, which was then incubated at 37 °C for gelatinization. HUVECs (5104 cells) in EGM-2 containing EGCG, cis- or trans-APDOEGCG at a concentration of 1 m M were added to each well; and the cells were incubated for 12 h at 37 °C. Photographs were taken with an Olympus IX71 microscope. Morphology Observation HUVECs (1104 cells/ chamber) were cultured in EGM-2 on a Lab-Tek® II Chamber SlideTM System (Thermo Fisher Scientific Inc.) for 24 h at 37 °C. After having been washed twice with Hank’s balance salt solution (HBSS), the cells were treated with EGCG, cis- or trans-APDOEGCG solution at a concentration of 1 m M for 24 h. Then, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and rendered permeable with 0.1% Triton X-100 solution. After having been washed with phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA) solution was added; and the cells were then incubated for 30 min at room temperature. Finally, the cells were incubated with 5 U/ml of rhodamine/ phalloidin (Molecular Probe®, Life Technologies, Eugene, OR, U.S.A.) and 10 m g/ml of 4,6-diamidino-2-phenylindole (DAPI, Invitrogen®, Life Technologies) in 1% BSA solution to stain F-actin or nucleus, respectively. The morphology of the HUVECs was observed under a confocal laser scanning microscope (LSM510META, Carl Zeiss). Intracellular Distribution of EGCG-TG in HUVECs HUVECs (1104 cells/chamber) were cultured in EGM-2 on a Lab-Tek® II Chamber Slide for 24 h at 37 °C. After having been washed twice with HBSS, the cells were incubated with EGCG-TG or with the original fluorescent probe TG as a control at a concentration of 1 m M for 0.5, 3, 6, 12, and 24 h. After having been fixed with paraformaldehyde, permeabilized, and blocked with BSA, the cells were incubated with 5 U/ml of rhodamine/phalloidin or 100 nM MitoTracker®Red CMXRos (Molecular Probe®, Life Technologies) for staining F-actin or mitochondria, respectively. Then the cell nuclei were stained with DAPI, and the distribution of EGCG-TG was observed under a confocal laser scanning microscope (LSM510META). The biodistribution of EGCG-TG was also observed after incubation of HUVECs with 10 m M EGCGTG for 6 h. Statistical Analysis Variance in a group was evaluated by using the F-test, and differences were evaluated by performing Student’s t-test. RESULTS Anti-proliferative Effect of EGCG Derivatives (APDOEGCG) on HUVECs We firstly examined the inhibitory effect of the synthesized EGCG derivatives, cis- or trans-APDOEGCG, on HUVEC proliferation, since our previous study demonstrated that the original EGCG inhibited HUVEC proliferation. As shown in Fig. 2A, EGCG and its derivatives effectively inhibited HUVEC proliferation when tested at more than 30 m M, and the anti-proliferative effect of

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Fig. 3. Capillary Tube Formation in the Presence of EGCG Derivatives Matrigel was diluted to 4 mg/ml with EGM-2 and added to the wells of a 24-well plate, and polymerization was allowed to occur. HUVECs (5104 cells/well) were added in each well in the presence or absence of 1 m M EGCG or its derivatives and incubated at 37 °C for 12 h. Capillary tube formation was observed under a microscope. Scale bars, 300 m m.

Fig. 2. Effect of EGCG Derivatives on HUVEC Proliferation, Motility, and Invasion (A) HUVECs (7500 cells/well) were cultured in EGM-2 with various concentrations of EGCG or EGCG derivatives for 48 h. Viable cells were determined by performing the TetracolorONE assay as described in Materials and Methods (n4). (B, C) HUVECs (5104 cells/300 m l) pre-labeled with BCECF-AM were added to the upper part of 8-m m-pored FluoroBlok inserts without (B) or with Matrigel-coating (C). A culture insert was then set on each well of a 24-well plate containing 700 m l EBM-2 per well, supplemented with 10% FBS. The indicated amount of EGCG or EGCG derivatives was then added to the upper well, and the cells were incubated for 24 h. After incubation, the number of cells that had invaded through the insert was counted under a fluorescence microscope (7 fields in each filter and 3 filters were determined in each point). Data represent average percentS.D. Significant differences from EGCG-treated group (∗ p 0.05; ∗∗∗ p0.001) and from the control (# p0.05; ### p0.001) are shown.

trans-APDOEGCG was the most potent among them. However, HUVEC proliferation was not affected at less than 10 m M EGCG or its derivatives. Since we wanted to elucidate the biological effect of EGCG, a concentration of EGCG or its derivatives at 1 m M was used for the following experiments. APDOEGCG-Mediated Inhibition of HUVEC Motility and Invasion Since migration of and invasion by vascular endothelial cells into the extracellular matrix are important steps for forming neovessels, we next examined the effect of the EGCG derivatives on HUVEC motility and invasion. Fluorescently labeled HUVECs were applied to the upper part of the FluoroBlok inserts in the absence or presence of Matrigel, and the number of cells that penetrated through to the lower side was counted. The motility assay (Fig. 2B) revealed that the EGCG derivatives inhibited the migration of the HUVECs at the concentration of 1 m M, whereas EGCG did not inhibit it at the same concentration. Furthermore, the inhibitory effect of trans-APDOEGCG was the highest among them. A similar effect was observed in the invasion

assay, in which trans-APDOEGCG significantly inhibited the cell invasion compared with non-derivatized EGCG (Fig. 2C). Since we previously reported that more than 10 m M EGCG was needed to strongly inhibit both motility of and invasion by HUVECs,13) the results obtained here indicate that derivatization of EGCG enhanced the anti-angiogenic activity of EGCG. Effect of EGCG Derivatives on Tube Formation in an in Vitro Angiogenesis Assay To confirm the anti-angiogenic effect of EGCG derivatives, we performed a tube formation assay with HUVECs, since HUVECs formed blood vessel-like capillaries on Matrigel. As shown in Fig. 3, both EGCG and EGCG-derivatives inhibited the capillary tube formation by HUVECs at even 1 m M; and the EGCG derivatives clearly inhibited it more so than did the EGCG. Effect of EGCG Derivatives on HUVEC Morphology It is possible that the inhibition of motility and invasion was due to morphological and cytoskeletal changes in HUVECs. To examine this possibility, we stained HUVECs with rhodamine/phalloidin and DAPI after the treatment with 1 m M EGCG or its derivatives and then observed them by confocal laser scanning microscopy. The confocal fluorescence images shown in Fig. 4 clearly demonstrate that the treatment with cis- or trans-APDOEGCG changed the distribution of Factin filament in the HUVECs and that the shape of the cells became narrow compared with the group of control and EGCG treatment, suggesting that EGCG derivatives changed the cellular morphology. This finding is consistent with our previous results showing that EGCG induces such morphological changes at a concentration as high as 50 m M.14) Observation of Distribution of EGCG Although candidates of target molecules of EGCG were previously reported, the distribution of EGCG after treatment has never been demonstrated due to the difficulty in specific detection of EGCG in certain cells. To resolve this difficulty, we synthesized fluorescence-labeled ()-cis-APDOEGCG derivative,

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Fig. 4. Morphological Changes in HUVECs by EGCG Derivatives HUVECs (1104 cells/chamber slide) were incubated with 1 m M EGCG or its derivatives at 37 °C for 24 h. Then, the cells were stained with 5 U/ml rhodamine/phalloidin followed by 10 m g/ml DAPI for 30 min at room temperature. F-actin (red colored) and nucleus (blue colored) were visualized under a confocal laser-scanning microscope. Scale bar, 20 m m.

Fig. 5. Intracellular Localization of EGCG-TG in HUVECs

EGCG-TG, by conjugating it to the fluorescence agent TokyoGreen (TG, Fig. 1), and examined the distribution of EGCG-TG in HUVECs. The confocal fluorescent images in Fig. 5A clearly show that most of the EGCG-TG fluorescence appeared as dots in the cytoplasm of HUVECs, suggesting that it was distributed in organelles. On the other hand, the fluorescence of free TG (green colored) was localized on the surface and in intracellular regions of the cells as a filamentous shape. Double staining with F-actin filament (red colored) indicated that free TG was co-localized with F-actin (orange-yellow colored). Fluorescent images also indicated that EGCG was incorporated into the cells within 3 h after the treatment. To determine the EGCG-localized organelle in HUVECs, the cells were stained with MitoTracker Red. The result indicated that at least a portion of EGCG-TG was localized in the mitochondria (Fig. 5B). DISCUSSION Many previous studies indicated that intake of tea catechins is associated with various biological activities such as anti-cancer, anti-obesity, anti-oxidative, anti-bacterial, and anti-viral ones and so on.22) We previously observed that 10 m M EGCG, which concentration did not affect the proliferation of HUVECs, significantly inhibited the invasion and tube formation of HUVECs in vitro and angiogenesis in vivo in dorsal air sac model mice. Interestingly, catechin, epicatechin, epicatechin-3-gallate, and epigallocatechin did not inhibit the motility and invasion of HUVECs at this concentration. For elucidation of the intracellular localization of EGCG,

(A) HUVECs (1104 cells/chamber slide) were incubated with 1 m M TokyoGreen (TG) or EGCG-TG at 37 °C for 0.5, 3, 6, 12 or 24 h. Then, the cells were stained with 5 U/ml rhodamine/phalloidin and 10 m g/ml DAPI for 30 min at room temperature. TG (green colored), F-actin (red colored), and nuclei (blue colored) were visualized by confocal laser-scanning microscopy. (B) HUVECs were incubated with 1 m M or 10 m M EGCG-TG for 6 h, and additionally incubated with MitoTracker Red and 10 m g/ml DAPI for 30 min at room temperature. EGCG-TG (green colored), mitochondria (red colored) and nuclei (blue colored) were visualized by confocal laser-scanning microscopy. Scale bar, 20 m m.

probe-conjugated EGCG such as fluorescence-conjugation is useful. We previously demonstrated that the phenolic hydroxyl groups on the A-ring in the flavan structure were not involved in the biological activity and that modification of the A-ring in the EGCG structure did not abolish but rather enhanced the functional ability of EGCG. Actually, the removal of the phenolic hydroxyl groups on the A-ring (5,7deoxyepigallocatechin gallate, DOEGCG) enhanced antiinfluenza infection activity.14) In the present study, we synthesized EGCG derivatives cis- or trans-APDOEGCG, in which linkers and reactive amino groups were grafted onto the Aring of EGCG. These derivatives would be expected to have several advantages: APDOEGCG enables the synthesis of fluorescence-labeled or other probe-conjugated EGCGs, and it might have stronger biological activity than EGCG, just as DOEGCG, since the linker moiety may endow the compound with enhanced hydrophobic interactions with biomembranes. In the present study, we examined the anti-angiogenic effect of cis- and trans-APDOEGCG in vitro by using HUVECs. In fact, both derivatives at a 1 m M concentration showed stronger inhibition of HUVEC motility, invasion, and tube formation than EGCG: These activities are shown for EGCG at 5 to 10 m M.21) Comparison between the 2 derivatives indicated that trans-APDOEGCG showed a bit stronger

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activity than cis-APDOEGCG. To explain this difference, we must look at the structural characteristics of cisAPDOEGCG and trans-APDOEGCG. cis-APDOEGCG is structurally similar to EGCG, whereas trans-APDOEGCG is not. It is possible that enantiomers display different biological activity. Smith and co-workers synthesized analogs of green tea polyphenols and found that analogs having characteristics similar to those of EGCG show stronger proteasome inhibition than those having characteristics different from EGCG.23) The present data are inconsistent with the previous results, however we do not know the reason at present. Next, we examined the intracellular distribution of EGCG by using TokyoGreen fluorescence-conjugated cis-APDOEGCG (EGCG-TG) in HUVECs. Previous report indicated that uptake of [H-3]EGCG by HT-29 cells was concentration-dependent without plateau and suggested that EGCG uptake mainly occurs through passive diffusion.24) Intracellular distribution of EGCG was also reported by Han and coworkers: They conjugated FITC to the galloyl moiety of EGCG, and observed the cytosolic and following nuclear distribution of fluorescence of this FITC-EGCG derivative in L929 cells.25) Since galloyl moiety of EGCG would be important for the EGCG function and unstable, we conjugated TG to the A-ring of EGCG in the present study. EGCG-TG was taken up into the HUVECs within 3 h; and the fluorescence showed a dotted pattern in the cytoplasm, indicating that it was localized in some cytoplasm organelle(s) of the cells. In contrast, free TokyoGreen was less incorporated into the cells; and a portion of it was co-localized with actin filaments. The result of performing an additional experiment using Mitotracker Red indicated that, at least a portion of the EGCG-TG interacted with the mitochondria. This observation is consistent with a previous report indicating that mitochondria of endothelial cells are an important target for inhibition of angiogenesis.26) Since modification of the A-ring, such as derivatization to APDOEGCG, did not change the mode of EGCG action, the distribution of EGCG-TG may reflect the actual EGCG localization: APDOEGCG suppressed motility and invasion of HUVECs and induced morphological changes similar to EGCG, although the concentration required was lower than that of EGCG. However, an alternative possibility that the fluorescence in mitochondria or in other cytoplasmic regions was derived from some degradation product(s) of EGCG-TG cannot be excluded at present. In conclusion, the EGCG analogs cis- and trans-APDOEGCG showed anti-angiogenic activity similar to but stronger than that of EGCG in vitro. The results of the intracellular distribution study using EGCG-TG suggested that EGCG was incorporated into HUVECs and that at least a portion of it was distributed into the mitochondria, suggesting that mitochondrial function was involved in the angiogenesis-inhibiting biological effect of EGCG. Acknowledgments The authors are thankful for the financial support to S. Piyaviriyakul (2009—2011) from the

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Department of Medical Services, Ministry of Public Health, Nonthaburi, Thailand. This work was also supported in part by a project of the Shizuoka Prefecture and Shizuoka City Collaboration of Regional Entities for the Advancement of Technological Excellence, by the Japan Science and Technology Agency (JST), by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and by the Global COE Program from MEXT, Japan. REFERENCES 1) Bhat T. A., Singh R. P., Food Chem. Toxicol., 46, 1334—1345 (2008). 2) Shimizu K., Oku N., Biol. Pharm. Bull., 27, 599—605 (2004). 3) Muñoz-Chápuli R., Quesada A. R., Medina M. Á., Cell. Mol. Life Sci., 61, 2224—2243 (2004). 4) Khan N., Mukhtar H., Cancer Lett., 269, 269—280 (2008). 5) Sharangi A. B., Food Res. Int., 42, 529—539 (2009). 6) Singh A. K., Seth P., Anthony, P., Husain M. M., Madhavan S., Mukhtar H., Maheshwari R. K., Arch. Biochem. Biophys., 401, 29—37 (2002). 7) Jung Y. D., Ellis L. M., Int. J. Exp. Path., 82, 309—316 (2001). 8) Shimizu K., Shimizu N. K., Hakamata W., Unno K., Asai T., Oku N., Biol. Pharm. Bull., 33, 117—121 (2010). 9) Tang F. Y., Nguyen N., Meydani M., Int. J. Cancer, 106, 871—878 (2003). 10) Rodriguez S. K., Guo W., Liu L., Band M. A., Paulson E. K., Meydani M., Int. J. Cancer, 118, 1635—1644 (2006). 11) Tachibana H., Koga K., Fujimura Y., Yamada K., Nat. Struct. Mol. Biol., 11, 380—381 (2004). 12) Umeda D., Yano S., Yamada K., Tachibana H., J. Biol. Chem., 283, 3050—3058 (2008). 13) Oku N., Matsukawa M., Yamakawa S., Asai T., Yahara S., Hashimoto F., Akizawa T., Biol. Pharm. Bull., 26, 1235—1238 (2003). 14) Yamakawa S., Asai T., Uchida T., Matsukawa M., Akizawa T., Oku N., Cancer Lett., 210, 47—55 (2004). 15) Furuta T., Hirooka Y., Abe A., Sugata Y., Ueda M., Murakami K., Suzuki T., Tanaka K., Kan T., Bioorg. Med. Chem. Lett., 17, 3095— 3098 (2007). 16) Aihara Y., Yoshida A., Furuta T., Wakimoto T., Akizawa T., Konishi M., Kan T., Bioorg. Med. Chem. Lett., 19, 4171—4174 (2009). 17) Ishii T., Mori T., Ichikawa T., Kaku M., Kusaka K., Uekusa Y., Akagawa M., Aihara Y., Furuta T., Wakimoto T., Kan T., Nakayama T., Bioorg. Med. Chem., 18, 4892—4896 (2010). 18) Yoshida A., Hirooka Y., Sugata Y., Nitta M., Manabe T., Ido S., Murakami K., Saha R. K., Suzuki T., Ohshima M., Yoshida A., Itoh K., Shimizu K., Oku N., Furuta T., Asakawa T., Wakimoto T., Kan T., Chem. Commun., in press. 19) Kobayashi T., UranoY., Kamiya M., Ueno T., Kojima H., Nagano T., J. Am. Chem. Soc., 129, 6696—6697 (2007). 20) Urano Y., Anal. Sci., 24, 51—53 (2008). 21) Yamakawa S., Furuyama Y., Oku N., Biol. Pharm. Bull., 23, 264—266 (2000). 22) Chacko S. M., Thambi P. T., Kuttan R., Nishigaki I., Chin. Med., 5, 13—21 (2010). 23) Smith D. M., Wang Z., Kazi A., Li L. H., Chan T. H., Dou Q. P., Mol. Med., 8, 382—392 (2002). 24) Hong J., Lu H., Meng X., Ryu J. H., Hara Y., Yang C. S., Cancer Res., 62, 7241—7246 (2002). 25) Han D. W., Matsumura K., Kim B., Hyon S. H., Bioorg. Med. Chem., 16, 9652—9659 (2008). 26) Park D., Dilda P. J., Mol. Aspects Med., 31, 113—131 (2010).