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Antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin isolated from Angelica keiskei roots through the inhibited activation and differentiation of M2 macrophages.

ABSTRACT

Background: Tumor growth and metastasis have been closely associated with the M2 macrophage-induced activation of tumor-associated macrophages (TAMs).

Purpose: The antitumor and antimetastatic actions of xanthangelol and 4-hydroxyderricin on the role of M2 macrophages in the TAMs of highly metastatic osteosarcoma LM8-bearing mice have not yet been fully elucidated. In order to clarify the mechanisms underlying the antitumor and antimetastatic actions of the above chalcones, we performed in vivo and in vitro studies.

Study Design: The antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin were examined in vivo and the effects on M2 macrophage differentiation and activation were examined in vitro.

Methods: We examined the antitumor and antimetastatic effects of xanthoangelol and 4-hydroxyderricin on highly metastatic osteosarcoma LM8-bearing mice (in vivo). Further, we examined their effects on the differentiation of interleukin (IL)-4 plus IL-13-induced M2 macrophages and activation of IL-4 plus IL13-induced M2 macrophages (in vitro). We also investigated the expression and phosphorylation of signal transducer and activator of transcript 3 (Stat 3) in the differentiation process of M2-polarized macrophages (in vitro).

Results: Xanthoangelol or 4-hydroxyderricin (25 or 50 mg/kg, twice daily) inhibited tumor growth, metastasis to the lung and liver, and TAM expression in tumors. In addition, xanthoangelol (10, 25 or 50 [micro]M) and 4-hydroxyderricin (5, 10, 25 or 50 [micro]M) inhibited the production of IL-10 and monocyte chemoattractant protein (MCP)-l in M2-polarized macrophages. This result indicated that xanthoangelol and 4-hydroxyderricin inhibited the activation of M2 macrophages. Furthermore, xanthoangelol (5-50 [micro]M) inhibited the phosphorylation of Stat 3 without affecting the expression of the Stat 3 protein in the differentiation process of M2 macrophages, which indicated that these chalcones inhibited the differentiation of M2 macrophages.

Conclusion: These findings suggested that the antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderrcin might be attributed to the regulated activated TAMs through the inhibition of activation and differentiation of M2 macrophages in the tumor microenvironment.

Keywords:

Chalcones

Antitumor

Antimetastatic

M2 macrophage

Stat3

Introduction

The roots of Angelica keiskei Koizumi (Umbelliferae) have been used as diuretic, laxative, analeptic, and lactagogue. Recent studies have reported that xanthoangelol [(E)-1-[3-[(E)-3,7-dimethylocta-2, 6-dien-1-yl]-2,4-dihydroxyphemyl]-3-(4-hydroxyphenyl)prop-2-en-1-one] and 4-hydroxyderrcin [(E)-1-[2-hydroxy-4-methoxy-3(3-methylbut-2-en-1-yl)phenyl]-3-(4-hydroxyphenyl) prop-2-en-1-one] (Fig. 1) isolated from A. keiskei roots exhibited anti-platelet actions (Son et al. 2014) as well as anti-inflammatory responses in RAW 264 macrophages (Yasuda et al, 2014) and human umbilical vein endothelial cells (Okura et al. 2011). It has been also reported that xanthoangelol and 4-hydroxyderricin inhibited the differentiation of preadipocytes to adipocytes (Zhang et al. 2013) and the biosynthesis of melanin (Arung et al. 2012). Furthermore, Akihisa et al. (2012) reported that the above chalcones displayed cytotoxic activities and antitumor-promoting effects. We previously reported that xanthoangelol and 4-hydroxyderricin exhibited antitumor and antimetastatic actions by inhibiting tumor-induced angiogenesis and/or the stimulation of immune function in Lewis lung carcinoma (LLC)-bearing mice (Kimura and Baba 2003; Kimura et al. 2004). Tumor-associated macrophages (TAMs) derived from circulating monocytes have been identified as the main components of the tumor microenvironment, and TAMs have consequently been shown to stimulate tumor growth and metastasis (Allavena and Mantovani 2012; Nakao et al. 2005; Schimieder et al. 2012). We also reported that tumor growth and metastasis may be stimulated by tumor-induced angiogenesis and lymphangiogenesis through increases in TAMs at the tumor site and blood monocyte chemoattractant protein-1 (MCP-1) (Kimura and Sumiyoshi 2013). This increase in TAMs around the tumor microenvironment has been closely associated with a poor prognosis in cancer patients (Joyce and Pollard 2009; Lewis and Pollard 2006; Sica et al. 2006, 2008). Macrophage phenotypes have been shown to express different receptors and produce different cytokines, and TAMs represent a subset of alternatively activated (M2) macrophages induced by [Th.sub.2] cytokines such as IL-4 and IL-13 (Gordon 2003; Joyce and Pollard 2009; Lewis and Pollard 2006; Mosser 2003; Mantovani et al. 2004; Sica et al. 2006, 2008). Therefore, M2 macrophages in TAMs accelerate tumor growth, invasion, and metastasis. However, the antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin on the role of M2 macrophages in the TAMs of highly metastatic osteosarcoma LM8-bearing mice have not yet been fully clarified. In the present study, we examined the effects of xanthoangelol and 4-hydroxyderricin on tumor growth and metastasis in osteosarcoma LM-8-bearing mice (in vivo), and on the differentiation and activation of M2 macrophages (in vitro).

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Materials and methods

Materials

Xanthoangelol and 4-hydroxyderricin (Baba et al. 1990; Kozawa et al., 1977, 1978) were supplied by Professor K. Baba (Department of Pharmacognosy, Osaka University of Pharmaceutical Sciences, Osaka, Japan). The purity of xanthoangelol and 4-hydroxyderricin was assessed by high performance liquid chromatography (HPLC) (Hitachi HPLC System, Hitachi, Tokyo, Japan) under the following two conditions; 1) The elution was performed with 56% acetonitrile containing 1% acetic acid (mobile phase) with a flow rate of 0.8 ml/min, monitoring wavelength at 330 nm, and Inertsil ODS-3 column (100 x 4.0 mm I.D.) (GL Science. Tokyo, Japan) (Fig. 2a); 2) The elution was performed with 70% methanol containing 1% acetic acid (mobile phase) with a flow rate of 0.75 ml/min, monitoring wavelength at 330 nm, and Inertsil ODS-3 column (100 x 4.0 mm l.D.) (Fig. 2b). The purity of xanthangelol and 4-hydroxyderricin was over 99.1% by the HPLC analysis using the above solvents (Fig. 2). Dulbecco's Modified Eagle's Medium (DMEM) and RPMI-1640 medium were obtained from Nissui Pharmacy Co. (Tokyo, Japan). Antibiotic and antimycotic solutions (100x) containing 10,000 units of penicillin, 10 mg/ml streptomycin, and 25 [micro]g/ml of amphotericin B in 0.9% NaCl solution were purchased from Sigma-Aldrich (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Gibco BRL (Auckland, New Zealand). One-hundred-millimeter culture dishes were purchased from Corning Glass Works (NY, USA). The human interleukin (IL)-10 and monocyte chemoattractant protein (MCP)-enzyme-linked immunosorbent assay (ELISA) kits were purchased from R & D Systems Inc. (MN, USA). Human recombinant IL-3 and IL-4 were purchased from R & D Systems Inc. The rabbit monoclonal anti-Stat 3 and rabbit monoclonal anti-phospho Stat 3 (Tyr 705) antibodies were purchased from Cell Signaling Technology Inc. (MA, USA). The mouse monoclonal anti-[beta]-action antibody was purchased from Sigma-Aldrich. The anti-F4/80 antibody was purchased from AbD Serotec (NC, USA). [gamma]-Cyclodextrin was purchased from Ensuiko Sugar Refinding Co. Ltd. (Yokohama, Japan). Cisplatin (CDDP) was supplied by Nihon Kayaku Co. (Tokyo, Japan) and dissolved in 0.9% NaCl. All chemicals used in this study were of reagent grade and purchased from Wako Pure Chemical Co. (Osaka. Japan).

Cells

Highly metastatic osteosarcoma LM8 cells and human monocyte THP-1 cells were obtained from the Institute of Development, Aging and Cancer, Tohoku University. LM8 cells were maintained in DMEM supplemented 10% FBS, penicillin (100 units/ml), streptomycin (100 [micro]/ml), and amphotericin (0.25 [micro]g/ml), and THP-1 cells were also maintained in RPMI-1640 medium containing the above FBS and antibiotics.

Animals

Male C3H/He mice (5 weeks old) were obtained from Japan SLC Co. (Shizuoka, Japan), housed for 1 week in a room with a controlled temperature at 25 [+ or -] 1[degrees]C and 60% relative humidity, and given free access to food and water during the experiments. Animal experiments were performed according to the Ethical Guidelines for Animal Experimentation by Ehime University and the Japanese Pharmacological Society, and the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The Animal Studies Committee of Ehime University approved the experimental protocol.

Tumor growth, tumor metastasis, and tumor histology in LM8-bearing mice (in vivo)

Solid-type LM8 was prepared by subcutaneous transplantation of 1 x [10.sup.5] cells into the backs of mice on day 0. Xanthoangelol and 4-hydroxyderricin were suspended in 5% [gamma]-cyclodextrin. Considering our previous reports in which the doses of xanthoangelol and 4-hydroxyderricin were 25-100 mg/kg body weight (oral administration) (Kimura and Baba 2003; Kimura et al. 2004), xanthoangelol and 4-hydroxyderricin (25 or 50 mg/kg body weight, twice daily) were administered orally for 30 days, starting 12 h after the implantation of tumor cells. CDDP (2.5 mg/kg) was administered intraperitoneally two times (Wednesday and thursday) per week to LM8-bearing (control) mice during the experimental period. Normal and control mice were orally administered 5% [gamma]-cyclodextrin solution alone according to the same schedule. Tumor volumes were determined every 2-3 days by direct measurements with calipers; volumes were calculated as the length x [width.sup.2]/2. On day 31, mice were killed with an overdose of diethylether, and the liver, lung, spleen, thymus, and tumors were quickly removed and weighed. Metastatic nodules in the liver, lung, or kidney were quantified with the following protocol: (a) the tumor metastasis of liver and lung were photographed; (b) the number of metastatic colonies of liver, lung, and kidney were counted independently by two of us using a stereoscopic microscope; (c) the tumor metastatic parts were cut to observe cut surface sites. All tumors and metastatic organs (liver and lung) were fixed in 10% buffered formalin for at least 24 h, progressively dehydrated in solutions containing an increasing percentage of ethanol (70, 80, 95, and 100%, v/v), cleared in Histoclear (FUME HOOD, AS-ONE, Tokyo, Japan), embedded in paraffin under a vacuum, and sectioned into 5 [micro]m-thick sections. After the paraffin-embedded tumor sections were deparaffinized, the expression of F4/80 (macrophage marker) in the tumors was determined by the immunoperoxidase technique using the anti-F4/80 antibody for the detection of macrophages. Four different microscopic fields were photographed per plate, and the number of F4/80-positive cells (macrophage marker) in the tumors was counted. After the paraffin-embedded metastatic organ sections of lung and liver were deparaffinized, their sections were stained with hematoxylin-eosin (HE). The metastatic tumor colonies in the lung and liver were photographed using a microscope (Leica DM3000S-1, Leica Microsystems, Tokyo, Japan).

Measurement of cytotoxicity in osteosarcoma LM8 cells and M2-polarized THP-1 macrophages

After LM8 cells (2 x [10.sup.4] cells/well) were cultured overnight in DMEM supplemented with 10% FBS, the medium was changed to fresh DMEM with 10% FBS, and the cells were exposed to the indicated amounts of xanthoangelol and 4-hydroxyderricin for 24 h. M2-polarized THP-1 macrophages were performed by the methods of Tjiu et al. (2009). Briefly, THP-1 monocytes (2 x [10.sup.4] cells/well) were cultured with 200 nM porbol 12-myristate 13 acetate (PMA) for 72 h in a 96-well microplate to induce differentiation. After the incubation period, the differentiated cells adhered to the culture plate and displayed morphological similarities to macrophages. To induce M2-polarized THP-1 macrophages, THP-1 macrophages differentiated by the above methods were cultured with 1L-4 (25 ng/ml) and IL-13 (25 ng/ml) for 24 h, and the differentiated M2 macrophages were then washed twice with Dulbecco's phosphate buffered saline (PBS) (pH 7.4). The differentiated M2 macrophages were cultured for 24 h with the indicated amounts of xanthangelol and 4-hydroxyderricin in fresh RPMI-1640 medium supplemented with 10% FBS. After the incubation period, the cytotoxicity against LM8 cells and THP-1 M2 macrophages was assessed using a cell counting kit (modified MTT assay, WST-1 assay; Wako Pure Chemical Co., Osaka, Japan) (Ishiyama et al. 1993).

Measurement of the production of IL-10 and MCP-1 in M2-polarized THP-1 macrophages

To induce M2-polarized THP-1 macrophages, THP-1 macrophages (5 x [10.sup.6] cells/well) differentiated by the above methods were cultured with IL-4 (25 ng/ml) and IL-13 (25 ng/ml) for 24 h in a 60mm culture dish, and the differentiated M2 macrophages were then washed twice with Dulbecco's phosphate buffered saline (PBS) (pH 7.4). The differentiated M2 macrophages were cultured with the indicated amounts of xanthoangelol and 4-hydroxyderricin for 24 h. After the incubation period, IL-10 and MCP-1 levels were measured in the medium using the human IL-10 and MCP-1 kits, respectively.

Measurement of the expression and phosphorylation of signal transducer and activator of transcription 3 (Stat3) in the differentiation process of M2-polarized THP-1 macrophages and THP-1 macrophages

PMA-treated THP-1 macrophages were cultured with IL-4 (25 ng/ml) and IL-13 (25 ng/ml) to differentiate M2 macrophages in the presence or absence of the above two chalcones for 24 h in a 60-mm culture dish. The cells were washed with ice-cold PBS (pH 7.0) and then lysed and centrifuged using the same methods described above. After being centrifuged, the supernatant was used to measure the phosphorylation of Stat 3 as well as its protein levels. Samples (80 [micro]g of protein) were subjected to electrophoresis on a 7.5% polyacrylamide gel, and the separated proteins were then transferred to blotting sheets of the polyvinylidene difluoride (PVDF) membrane (Bio-Rad Lab., CA, USA). The blot was incubated with 5% skimmed milk to bind on the PVDF membrane. The solution of the anti-Stat 3 rabbit, anti-phospho Stat 3 rabbit monoclonal, or anti-[beta]-actin mouse monoclonal antibody was added to bind to their respective specific proteins (primary antibody reaction), and the alkaline phosphatase-conjugated anti-rabbit IgG goat antibody (MP Biomedicals, Tokyo, Japan) or alkaline phosphatase-conjugated anti-mouse IgG goat antibody (Sigma, Tokyo, Japan) was then added to detect these proteins (secondary antibody reaction). The location of the respective antibody was detected by 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (BCIP/NBT) solution (Sigma, Tokyo, Japan).

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Statistical analysis

All values are expressed as means [+ or -] S.E.M. When data followed a normal distribution, data were analyzed by a one-way ANOVA or repeated-measures ANOVA. When the F-test was significant, means were compared using the Turkey-Kramer or Dunnett test with Stat View (SAS institute Inc., Tokyo, Japan). Differences were considered significant at P < 0.05.

Results

Cytotoxicity of xanthangelol and 4-hydroxyderricin in LM8 cells and M2-polarized THP-1 macrophages (in vitro)

Xanthoangelol and 4-hydroxyderricin showed 32 [+ or -] 1 and 33 [+ or -] 1% cytoxicity at 50 [micro]M, and 54 [+ or -] 2 and 45 [+ or -] 2% cytotoxicity at 100 [micro]M, against LM8 cells, respectively. Furthermore, xanthoangelol and 4-hyderoxyderricin also showed cytotoxicity against THP-1-induced M2 macrophages as follows: The percentage of cytoxicity was 21 [+ or -] 1 and 25 [+ or -] 1% at 50 [micro]M, and 33 [+ or -] 2 and 36 [+ or -] 2% at 100 [micro]M. Both chalcones had no cytotoxic effect on LM8 cells and M2 macrophages at concentrations of 5-25 [micro]M (data not shown).

Antitumor actions of xanthoangelol and 4-hydroxyderricin in LM8-bearing mice (in vivo)

Xanthoangelol (25 and 50 mg/kg, twice daily) significantly inhibited tumor growth on days 13-30 (Fig. 3a). Final tumor weights were also significantly decreased by xanthoangelol (25 and 50 mg/kg, twice daily) (Fig. 3b). Immunohistochemical observations revealed that xanthoangelol (25 and 50 mg/kg, twice daily) reduced the expression of F4/80 (a marker of macrophage) in the solid tumors of mice with subcutaneously implanted LM8 cells (Fig. 3e and Table 1). Although 4-hydroxyderricin inhibited tumor growth on days 13-30 (Fig. 3c), it did not significantly affect final tumor weights (Fig. 3d). The expression of F4/80 was reduced by the oral administration of 4-hydroxyderricin (50 mg/kg, twice daily) (Fig. 3e and Table 1). Anti-cancer drug CDDP (2.5 mg/kg, 2 times/week, ip) inhibited tumor growth and diminished tumor weights (Fig. 3a-d).

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Antimetastatic actions of xanthoangelol, and 4-hydroxyderricin in LM8-bearing mice (in vivo)

Xanthoangelol (25 and 50 mg/kg, twice daily) and 4-hydroxyderricin (50 mg/kg, twice daily) significantly inhibited lung metastasis, and they also inhibited the increases in lung weights due to tumor metastasis (Figs. 4a-d and 5a). Anti-cancer drug CDDP also inhibited the lung metastasis and the increases in lung weights (Figs. 4a-d and 5a). Furthermore, xanthoangelol and 4-hydroxyderricin inhibited liver metastasis at doses of 25 and 50 mg/kg (Fig. 5b and Table 2).

Effects of xanthoangelol, and 4-hydroxyderricin on body weights, food consumption, and various weights in LM8-bearing mice (in vivo)

As shown in Table 3, no significant differences were observed in the final body weights, food consumption, or weights of the liver, spleen, and thymus between normal, vehicle-treated LM8-bearing mice (control), xanthoangelol-, and 4-hydroxyderricin-treated LM8-bearing mice. On the other hand, anti-cancer drug CDDP caused severe adverse effects such as the reduction in final body, spleen and thymus weights, and food consumption compared to normal or control mice. Thus, in contrast to anti-cancer drug CDDP, xanthoangelol and 4-hydroxyderricin did not cause severe adverse reactions.

Effects of xanthoangelol and 4-hydroxyderricin on the production of IL-10 and MCP-1 in M2-polarized THP-1 macrophages (in vitro)

The production of IL-10 and MCP-1 was significantly increased by the differentiation of IL-4- plus IL-13-induced M2-polarized THP-1 macrophages (M2-macrophages) from PMA-treated THP-1 macrophages. Xanthoangelol inhibited the production of IL-10 and MCP-1 in IL-4- plus IL-13-induced M2 macrophages at concentrations of 10, 25, and 50 [micro]M (Fig. 6a and b). 4-Hydroxyderricin also inhibited the production of IL-10 at a concentration of 50 [micro]M as well as that of MCP-1 at 5-50 [micro]M in M2 macrophages (Fig. 6c and d).

Effects of xanthoangelol and 4-hydroxyderricin on the expression of the Stat3 protein and Stat3 phosphorylation (p-Stat3) in the differentiation process of M2-polarized THP-1 macrophages (in vitro)

The phosphorylation of the Stat3 protein was stimulated by the differentiation of IL-4- plus IL-13-induced M2-polarized macrophages; however, no significant difference was observed in the expression of the Stat3 protein between PMA-treated THP-1 macrophages and IL-4- plus IL-13-induced THP-1 M2-polarized macrophages. Neither xanthoangelol nor 4-hydroxyderricin had an effect on the expression of the Stat3 protein in the differentiation process of M2 macrophages (Fig. 7a, b, d, and e). The phosphorylation of the Stat3 protein was inhibited by xanthoangelol and 4-hydroxyderricin at concentrations of 10-50 [micro]M (Fig. 7a, c, d, and f).

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Discussion

We previously demonstrated that xanthoangelol and 4-hydroxyderricin isolated from A. keiskei roots exhibited antitumor and antimetastatic actions by inhibiting tumor-induced angiogenesis (Kimura and Baba 2003; Kimura et al. 2004). Although we could not isolate the TAMs from tumors of tumor-bearing mice treated with xanthoangelol and 4-hydroxyderricin, these chalcones reduced the number of macrophages (F4/80 expression) in the tumors collected from highly metastatic osteosarcoma LM8-bearing mice. Macrophages have been divided into classically activated macrophages (Ml-polarized macrophages) and alternatively activated macrophages (M2-polarized macrophages) based on their abilities to produce T helper type 1 ([Th.sub.1] and [Th.sub.2] cytokines, respectively (Mantovani et al. 2004). M2 macrophages have high 1L-10 and low 1L-12 levels, and produce several growth factors such as vascular endothelial growth factor (VEGF) (Gordon 2003; Mosser 2003). Xanthoangelol and 4-hydroxyderricin inhibited the activation of M2 macrophages by inhibiting the production of IL-10 and MCP-1 in M2-polarized macrophages. Stat 3 has been closely associated with the tumor microenvironment as well as tumor growth and metastasis, and its signaling in macrophages has been linked to the regulation of immunosuppression and angiogenesis (Matsukawa et al. 2003; Takeda et al. 1999; Yu et al. 2007). Sica and Bronte (2007) reported that the activation of Stat 3 was essential for the differentiation of M2 macrophages. We herein demonstrated that xanthoangelol and 4-hydroxyderricin inhibited the differentiation process of M2 macrophages by inhibiting the phosphorylation of Stat3 without affecting its expression in the differentiation of IL-4-plus IL-13-induced M2 macrophages. These results suggested that the antitumor and/or antimetastatic actions of xanthoangelol and/or 4-hydroxyderricin were due to the inhibited differentiation of M2 macrophages caused by a decrease in the phosphorylation of Stat 3 and inhibited activation of M2 macrophages due to lower levels of IL-10 and MCP-1 being produced in M2 macrophages. It appears that the anti-angiogenic and immunomodulatory effects of xanthoangelol and 4-hydroxyderricin in LLC-bearing mice (Kimura and Baba 2003; Kimura et al. 2004) may also be due to the inhibition of activation and differentiation of M2 macrophage. Xanthoangelol and 4-hydroxyderricin showed cytotoxicity against LM8 cells and M2 macrophages, at the concentrations of 50 and 100 [micro]M. Although the above results might have partly contributed to antitumor and antimetastatic actions of xanthoangelol and 4-hydroxyderricin, further studies are needed to clarify the relationship between in vitro and in vivo experiments. In the in vivo study, the antitumor action of xanthoangelol is higher than that of 4-hydroxyderricin. One or both structural features (presence of a 4'-free phenolic OH and presence of a longer isoprene moiety in C-3') could be the cause of the better activity of xanthoangelol respective of 4-hydroxyderricin, correlation that should be elucidated in further works.

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Conclusions

This is the first report showing that natural chalcones xanthoangelol and 4-hydroxyderricin exhibit antitumor and antimetastatic action through the inhibition of M2 macrophage activation and differentiation in the tumor microenvironment.

Conflict of interest

Authors declare that they have not any conflict of interest.

Abbreviations: CDDP, cisplatin; HE, hematoxylin-eosin; IL, interleukin; LLC, Iewis lung carcinoma; MCP, monocyte chemoattractant protein; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; Stat 3, signal transducer and activator of transcription 3; TAM, tumor-associated macrophage.

ARTICLE INFO

Article history:

Received 21 October 2014

Revised 8 May 2015

Accepted 13 May 2015

This paper is dedicated to Dr. Maho Sumiyoshi, 42 years old, who passed away on December 11th, 2014,50 days after the original submission of this article.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 26460908 to Yoshiyuki Kimura) from the Ministry of Education, Culture, Sports, Science, and Technology. Dr. Y. Kimura designed the experiments, conducted all the experimental work, wrote the manuscript, and discussed it with Dr. M. Sumiyoshi; Dr. M. Sumiyoshi performed all the experimental analyses and helped in writing the manuscript; and Dr. K. Baba and Dr. M. Taniguchi carried out the isolation of the two chalcones (xanthoangelol and 4-hydroxyderricin) from Angelica keiskei roots and performed the HPLC analyses. All authors read and approved the final manuscript. We wish to express our post mortem acknowledgement to Dr. Maho Sumiyoshi, first author of this work, for her great commitment to carrying out the tests, the discussion of the results and the writing of the text, thus in all over the process of preparing this work.

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Maho Sumiyoshi (a), Masahiko Taniguchi (b), Kimiye Baba (b), Yoshiyuki Kimura (c), *

(a) Division of Functional Histology, Department of Functional Biomedicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan

(b) Department of Pharmacognosy, Osaka University of Pharmaceutical Sciences, Takatsuki City, Osaka 569-1094, Japan

(c) Division of Biochemical Pharmacology, Department of Basic Medical Research, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan

* Corresponding author. Tel: +81 89 960 5922; fax: +81 89 960 5239.

E-mail address: yokim@rn.ehime-u.ac.jp (Y. Kimura).

http://dx.doi.org/10.1016/j.phymed.2015.05.005
Table 1
Effects of xanthoangelol and 4-hydroxyderricin on the number of F4/80
(macrophage marker)-positive cells in tumors collected from
LM8-bearing C3H/He male mice.

                             Animal no.   Macrophage number/field

LM8-bearing mice (control)   8            215 [+ or -] 33
+ Xanthoangelol
  (25 mg/kg, twice daily)    7            101 [+ or -] 20 *
  (50 mg/kg, twice daily)    7             60 [+ or -] 15 *
+ 4-Hydroxyderricin
  (25 mg/kg, twice daily)    7            167 [+ or -] 40
  (50 mg/kg, twice daily)    7            111 [+ or -] 37 *

Values are expressed as the mean [+ or -] SE for 7-8 mice.

* Significantly different from LM8-bearing mice (control), P < 0.05.

Table 2
Effects of xanthoangelol, 4-hydroxyderricin and anti-cancer drug CDDP
on metastasis to the liver, lung, and kidney in LM8-bearing C3H/He
male mice.

                                   Liver       Lung        Kidney
                                   metastasis  metastasis  metastasis

LM8-bearing mice (control)         9/15        15/15       8/15
                                   (60.0%)     (100.0%)    (53.3%)
+ Xanthoangelol                    2/7         1/7         0/7
  (25 mg/kg, twice daily)          (28.6%)     (14.3%)     (0%)
                                   1/7         2/7         0/7
  (50 mg/kg, twice daily)          (14.3%)     (28.6%)     (0%)
+ 4-Hydroxyderricin                0/7         3/7         0/7
  (25 mg/kg, twice daily)          (0%)        (42.9%)     (0%)
                                   0/7         2/7         0/7
  (50 mg/kg, twice daily)          (0%)        (28.6%)     (0%)
+ CDDP (2.5 mg/kg, 2 times/week)   2/7         3/7         0/7
                                   (28.6%)     (42.9%)     (0%)

Values are expressed as the number of metastatic mice/all mice.

Table 3
Effects of xanthoangelol, 4-hydroxyderricin and anti-cancer drug CDDP
on body weights, food intake, and various tissue weights in
LM8-bearing C3H/He male mice.

                                  Final body weight (g)

Normal (n = 14)                   23.80 [+ or -] 0.32
LM8-bearing mice
(Control) (n = 15)                21.90 [+ or -] 0.34
+ Xanthoangelol
  (25 mg/kg x 2/day) (n = 7)      22.10 [+ or -] 0.44
  (50 mg/kg x 2/day) (n = 7)      22.40 [+ or -] 0.54
+ 4-Hydroxyderricin
  (25 mg/kg x 2/day) (n = 7)      21.10 [+ or -] 0.28
  (50 mg/kg x 2/day) (n = 7)      21.30 [+ or -] 0.64
+ CDDP
  (2.5 mg/kg x 2/week) (n = 7)    17.40 [+ or -] 0.81 *

                                  Food intake
                                  (g/mouse/30 day)

Normal (n = 14)                   2.53 [+ or -] 0.04
LM8-bearing mice
(Control) (n = 15)                2.41 [+ or -] 0.04
+ Xanthoangelol
  (25 mg/kg x 2/day) (n = 7)      2.42 [+ or -] 0.06
  (50 mg/kg x 2/day) (n = 7)      2.47 [+ or -] 0.06
+ 4-Hydroxyderricin
  (25 mg/kg x 2/day) (n = 7)      2.31 [+ or -] 0.05
  (50 mg/kg x 2/day) (n = 7)      2.35 [+ or -] 0.05
+ CDDP
  (2.5 mg/kg x 2/week) (n = 7)    1.88 [+ or -] 0.09 *

                                  Liver (mg)

Normal (n = 14)                   1241.80 [+ or -] 22.36
LM8-bearing mice
(Control) (n = 15)                1086.70 [+ or -] 74.83
+ Xanthoangelol
  (25 mg/kg x 2/day) (n = 7)      1334.90 [+ or -] 64.46
  (50 mg/kg x 2/day) (n = 7)      1296.30 [+ or -] 105.07
+ 4-Hydroxyderricin
  (25 mg/kg x 2/day) (n = 7)      1107.40 [+ or -] 83.40
  (50 mg/kg x 2/day) (n = 7)      1237.30 [+ or -] 46.56
+ CDDP
  (2.5 mg/kg x 2/week) (n = 7)     789.90 [+ or -] 41.21#

                                  Spleen (mg)

Normal (n = 14)                   63.70 [+ or -] 1.26
LM8-bearing mice
(Control) (n = 15)                70.30 [+ or -] 4.15
+ Xanthoangelol
  (25 mg/kg x 2/day) (n = 7)      65.70 [+ or -] 3.73
  (50 mg/kg x 2/day) (n = 7)      74.90 [+ or -] 4.25
+ 4-Hydroxyderricin
  (25 mg/kg x 2/day) (n = 7)      72.00 [+ or -] 6.20
  (50 mg/kg x 2/day) (n = 7)      63.00 [+ or -] 3.29
+ CDDP
  (2.5 mg/kg x 2/week) (n = 7)    40.70 [+ or -] 5.31 *#

                                  Thymus (mg)

Normal (n = 14)                   25.50 [+ or -] 0.96
LM8-bearing mice
(Control) (n = 15)                23.50 [+ or -] 1.96
+ Xanthoangelol
  (25 mg/kg x 2/day) (n = 7)      23.00 [+ or -] 1.82
  (50 mg/kg x 2/day) (n = 7)      20.40 [+ or -] 3.36
+ 4-Hydroxyderricin
  (25 mg/kg x 2/day) (n = 7)      25.10 [+ or -] 2.97
  (50 mg/kg x 2/day) (n = 7)      23.50 [+ or -] 3.56
+ CDDP
  (2.5 mg/kg x 2/week) (n = 7)     4.29 [+ or -] 2.41 *#

Values are expresses as the mean [+ or -] S.E.M. for 7-15 mice; n:
animal number.

# Significantly different from normal mice.

* Significantly different from LM8-bearing mice (control), P < 0.05.
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Author:Sumiyoshi, Maho; Taniguchi, Masahiko; Baba, Kimiye; Kimura, Yoshiyuki
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jul 15, 2015
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