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Macrophage-oriented cytotoxic activity of novel triterpene saponins extracted from roots of Securidaca inappendiculata
International Immunopharmacology 1 Ž2001. 1989–2000 www.elsevier.comrlocaterintimp
Macrophage-oriented cytotoxic activity of novel triterpene saponins extracted from roots of Securidaca inappendiculata Satoru Yui a,) , Kazuyoshi Ubukata a , Kazumi Hodono a , Mikio Kitahara b, Yoshihiro Mimaki c , Minpei Kuroda c , Yutaka Sashida c , Masatoshi Yamazaki a a
Faculty of Pharmaceutical Sciences, Teikyo UniÕersity, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan Takasago Research Laboratories, Research Institute, Kaneka Corporation, Miyamae-cho, Takasago-cho, Takasago, Hyogo 676-8688, Japan Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo UniÕersity of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan b
c
Received 1 March 2001; received in revised form 31 May 2001; accepted 27 June 2001
Abstract It is recognized that macrophages in peripheral tissues often proliferate under pathological conditions such as tumors, inflammation and atherosclerosis. Because the growth state of macrophages is believed to be a factor regulating the pathological process of the diseases, substances that regulate macrophage growth or survival may be useful for disease control. In this paper, we identified the activity inhibiting macrophage growth in a hot water extract of roots of Securidaca inappendiculata. The extract markedly inhibited macrophage colony-stimulating factor ŽM-CSFrCSF-1.-induced growth of macrophages, whereas it exerted a less potent effect on growth of Concanavalin A ŽCon A.-stimulated thymocytes or M-CSF-stimulated bone marrow cells. The inhibition of macrophage growth was caused by a cytotoxic effect rather than a cytostatic effect. Cell death was due to the induction of apoptosis, as judged by staining with terminal deoxynucleotidyl transferase-mediated d-UTP nick end labelling ŽTUNEL.. The cytotoxic activity seemed to be specific to peripheral macrophages; it showed a weak effect on the growth and survival of tumor cell lines including a macrophage-like cell line, J-774.1. Moreover, the saponin fraction induced apoptotic cell death of macrophages only when they were stimulated by M-CSF; it did not affect the viability of macrophages cultured without M-CSF or with granulocytermacrophage-CSF. We determined the structures of the two active triterpene saponin compounds in the fraction, named securioside A and securioside B having a 3,4-dimethoxycinnamic group which is essential for the cell death-inducing activity. They are believed to be the primary compounds of new drugs for the treatment of pathological states in which macrophage proliferation occurs. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Macrophages; Apoptosis; M-CSF; Saponin compounds
1. Introduction
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Corresponding author. Tel.: q81-426-85-3736; fax: q81-42685-2574. E-mail address: [email protected] ŽS. Yui..
It is widely recognized that macrophages play a central role in the regulation of inflammatory and immunological reactions through various functions including phagocytic elimination of foreign or dena-
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 2 6 - 6
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tured substances, and secretion of various cytokines or reactive oxygen intermediates w1,2x. Under local pathological conditions, the function of macrophages depends on qualitative and quantitative aspects, namely, their activation state and their accumulation in local sites. There are cumulative observations of the peripheral proliferation of macrophages in pathological tissues such as tumors w3,4x, atherosclerotic lesions w5–8x and several inflammatory sites w9–12x. The protein factors, macrophage colony-stimulating factor ŽM-CSFrCSF-1. and granulocytermacrophage colony-stimulating factor ŽGM-CSF. are growth factors for peripheral macrophages w13–15x. In addition to them, we reported that materials to be scavenged by macrophages via scavenger receptorŽs. induce macrophage growth in the mediation of GMCSF w16–18x. The normally undividing macrophages may respond to the above factors and enter a cell division cycle under pathological conditions. The local proliferation is thought to contribute to macrophage accumulation, and it may also influence the pathological process. For example, in atherosclerotic lesions, the proliferation of lipid-laden macrophages that scavenge oxidized low density lipoprotein ŽLDL. may be implicated in the accumulation of cholesteryl esters in the intima of blood vessels, as well as in the formation of atherosclerotic plaques w5–7,16,19x. Under many other inflammatory conditions, macrophage expansion in situ is believed to influence the consequence of a disease w9–12x since, as described above, macrophage is located in a central position of inflammation regulation. Accordingly, how to inhibit macrophage proliferation andror eliminate macrophages in the mitotic stage is an important aspect of the regulation of inflammatory diseases. To identify a substance that inhibits macrophage proliferation, we performed a screening study among hot water extracts of plant materials that have been traditionally used as Chinese medicines and are known to have anti-inflammatory activity. Among these materials, we found that the extract of the roots of Securidaca inappendiculata contains saponin compounds that induce cytotoxic activity against MCSF-stimulated macrophages. We identified novel triterpene saponins as cytotoxic molecules against macrophages and they were named securioside A and securiosides B w20x. In this report, we further
extended the characterization of the cytotoxic activity of these saponins against macrophages.
2. Materials and methods 2.1. Reagents Soyasaponin and glycyrrhizin were purchased from Wako ŽPure Chemicals, Osaka, Japan.. Concanavalin A ŽCon A. was from E.Y. Laboratories ŽSan Mateo, CA.. Recombinant mouse M-CSF and GM-CSF were purchased from Genzyme ŽCambridge, MA.. 2.2. Mice Male C3HrHe mice and C3HrHeJ mice were purchased from breeding colonies in Japan SLC ŽShizuoka, Japan.. 2.3. Macrophage culture Peritoneal cells were obtained 3–5 days after i.p. injection of 30 mg of starch into each C3HrHe mouse. These cells were suspended in RPMI 1640 medium ŽNissui Seiyaku, Tokyo, Japan. supplemented with 5% heated-inactivated fetal calf serum ŽFCS, Summit, Ft. Collins, CO, USA., penicillin Ž100 Urml. and Kanamycin Ž60 m grml.. For wmethyl3 Hx-thymidine Žw3 Hx-TdR. incorporation assay and MTT assay, the peritoneal cells were incubated in 96-well microplates ŽCorning, Corning, NY. at 2 = 10 4 cellsrwell. For morphological observation, these cells were incubated in 24-well microplates ŽNunc, OK-4000, Roskilde, Denmark. at 0.5 = 10 5 cellsrwell. The cells were incubated for 90 min at 37 8C in a CO 2-incubator to allow them to adhere to the culture plates. The medium was then removed, and the nonadherent cells were removed by three vigorous washings with prewarmed PBS solution. More than 95% of the adherent cells were judged to be macrophage both by Giemsa staining and carbon particle uptake. These macrophages were added with the sample to be tested and simultaneously with L-cell-conditioned medium that was used as crude M-CSF w13x. The cells were cultured at 37 8C in a humidified atmosphere of 5% CO 2 in air
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with 0.2 ml Žfor 96 well plates. or 1 ml Žfor 24 well plates. of medium without changing the medium. 2.4. [3H]-TdR incorporation The incorporation of 3 H-TdR into cultured macrophages was measured as described w18x. Briefly, after the indicated culture periods, w3 Hx-TdR Ž2960 TBqr mmol, New England Nuclear, Boston, MA. was added to each plate at 37 kBqrml and incubated for 18 h. The medium was discarded, and the cells were dissolved in 100 m l of 0.5% sodium dodecyl sulfate, followed by addition of 100 m l of cold 10% trichloroacetic acid. The resulting trichloroacetic acidinsoluble material was collected on filters with Labo mash LM-101 ŽFutaba Medical, Tokyo.. The filters were dried, and their radioactivity was counted in a liquid scintillation spectrophotometer. All experiments were performed in triplicate. 2.5. MTT assay Macrophage cell death was evaluated by MTT assay w21x. Macrophages in 96 well plates were added with 25 m lrwell of 3-Ž4,5-dimetyl-2-thiazolyl.-2,5-diphenyl-2 H-tetrazolium bromide ŽMTT, 5 mgrml. and plates were incubated for an additional 3 h. Then, 150 m l of the supernatants was discarded, 100 m l of acid-isopropanol solution Ž0.04 N HCl in 2-propanol. was added to each well and the optical density Ž588 nm. was measured with a microplate reader ŽMTP-100, Corona Electric, Ibaragi, Japan.. Experiments were performed with duplicate determinations and results were expressed as relative values Ž% of control. in which each mean value was compared with that of medium alone. 2.6. Microscopic obserÕation Morphological observations of cultured macrophages were made by inverted, phase-contrast microscopy. DNA breaks in cultured macrophages were detected in situ by the terminal deoxynucleotidyl transferase ŽTdT.-mediated dUTP nick end labeling ŽTUNEL. method, which was originally described by Gavrieli et al. w22x, using a kit ŽIn Situ Cell Death Detection Kit, fluorescein, Boeringer-Mannheim,
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Mannheim, FRG.. The TUNEL reaction enzymatically labels free 3X-OH termini of DNA strand breaks with fluorescein-dUTP with TdT. The fluorescein incorporated into cells was observed by fluorescence microscopy. 2.7. Other cells Bone marrow cells were collected from femurs of C3HrHe mice, and cultured in the presence of 10% L-cell-conditioned medium. 3 H-TdR was pulsed on day 3 for 16 h and cells were harvested as described in macrophage culture. Thymocytes were obtained from C3HrHeJ mice. The cells Ž1.5 = 10 6 cellsr200 m l. were cultured in wells of 96-well plates with 5 m grml of Con A. 3 H-TdR was pulsed on day 2 for 5 h and cells were immediately trapped in glass filters using water as a washing solution. The mouse fibrosarcoma line, L-929, was maintained in MEM supplemented with 5% calf serum. The macrophagelike cell line, J774.1 obtained from HSRRB Cell Bank ŽOsaka, Japan. and EL-4 mouse thymoma were maintained in RPMI 1640 medium supplemented with 5% FCS. Human dermal fibroblasts were purchased from Cell Systems ŽKirkland, WA. and were maintained in RPMI 1640 medium supplemented with 10% FCS. These tumor cell lines and human dermal fibroblasts were cultured in wells of 96-well plates at the concentration of 1 = 10 4 cellsrwell, respectively. 2.8. Plant materials The plant materials Ž59 species. used for screening study were from the sources listed elsewhere, in which we had screened the inhibitory substance for calprotectin-induced apoptosis w23x. 2.9. Purification of the substances inducing macrophage cell death The dried roots of S. inappendiculata Hassk. Ž154 g. were extracted with distilled water Žtotal 3000 ml. at 110 8C for 3 h. After the residue was removed by centrifugation, the supernatant was lyophilized, and 8.85-g dried powder was obtained. A part of the dried extract was dissolved in water and was partitioned into aqueous phase and the ether fraction
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using extraction with diethyl ether. The aqueous phase was thereafter extracted with butanol to produce the butanol fraction and the resultant aqueous fraction. Each fraction was evaporated and dissolved in MeOH. In a pilot experiment, 120 mg of hot water extract yielded 16 mg of ether fraction, 24 mg of butanol fraction and 47 mg of aqueous fraction, respectively. The butanol fraction, termed Fr. B Ž500 mg from an experiment on a larger scale. was further purified with Wako-gel C-200 column chromatography ŽWako.. After elution of CHCl 3rMeOH Ž3:1, vrv., which resulted in a 148-mg residue after evaporation, the fraction including cytotoxic activity against macrophages was eluted with MeOH. Two purified saponins, namely, securioside A and securioside B and their deacylated forms were prepared from the MeOH eluate as described elsewhere w20x. The LPS content of the MeOH eluate fraction, securioside A and securioside B was measured by Toxicolor ŽSeikagaku, Tokyo, Japan., and was estimated to be 2.0, 3.4 and 2.5 ngrmg, respectively.
3. Results 3.1. Screening of hot water extracts of plant materials to identify substances inhibiting macrophage growth To find substances having inhibitory activity for macrophage growth, we screened various hot water extracts of 59 plant species that have been used as Chinese medicine w23x. The extracts were added to macrophage cultures in the presence of L-cell-conditioned medium as a macrophage growth factor. Three
Fig. 1. Inhibitory activities of the hot water extract from roots of S. inappendiculata against 3 H-TdR incorporation of macrophages, thymocytes and bone marrow cells. ŽA. Starch-induced macrophages were cultured with the indicated concentrations of hot water extract in the presence of 20% L-cell-conditioned medium for 6 days. ŽB. Thymocytes were cultured with the indicated concentrations of hot water extract in the presence of Con A for 2 days. ŽC. Bone marrow cells were cultured with the indicated concentrations of hot water extract in the presence of 10% L-cell-conditioned medium for 3 days. 3 H-TdR incorporation into these cells was measured as described in Materials and Methods. Bars represent S.D.
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of these extracts Ž S. inappendiculata, Cimicifuga foetida and Arctium lappa. showed strong inhibitory activity Ždata not shown.. In this paper, we studied the properties of the substances present in S. inappendiculata. Fig. 1A depicts the dose–response relationship of the inhibitory effect of the crude extract of S. inappendiculata on macrophage 3 H-TdR incorporation; the inhibition was seen from 0.63 m grml. To access the specificity of the activity of this extract, we examined its effects on bone marrow cells and thymocytes. As shown in Fig. 1B and C, only a high concentration of the extract Ž40 m grml. reduced 3 H-TdR incorporation into thymocytes or bone marrow cells which were stimulated with Con A or M-CSF, respectively, suggesting that macrophages are specifically sensitive to the substanceŽs. included in S. inappendiculata.
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3.2. Partial purification of the actiÕe substances To purify the active substances, we performed fractionation of the extract by sequential extraction using diethyl ether and then butanol. As shown in Fig. 2A, the butanol extract ŽFr. B. showed higher specific activity than the crude extract, while the aqueous fraction and ether fraction showed weaker activity than the crude extract. To ascertain whether the inhibition is due to the cytostatic effect or the cytocidal effect, the morphology of macrophages cultured without or with Fr. B is shown in Fig. 2C—a and b, respectively. More than half of the cells cultured with Fr. B for 2 days showed small, shrunken morphology. Although some damaged cells still adhered to the culture substrate, they were no longer spreading. The breb formation
Fig. 2. Macrophage growth-inhibiting or toxic activity of partially purified samples partitioned from the extract of S. inappendiculata. ŽA. Starch-induced macrophages were cultured with the following samples obtained by organic solvent extraction of the hot water extract in the presence of 20% L-cell-conditioned medium. Žv . Crude hot water extract, Ž`. ether-extracted fraction, Ž^. butanol fraction ŽFr. B., ŽI. aqueous fraction. 3 H-TdR incorporation into macrophages was assayed on day 6. ŽB. Starch-induced macrophages were cultured with the samples obtained by silicic column chromatography of Fr. B in the presence of 20% L-cell-conditioned medium: Ž^. Fr. B, Že. the eluate with CHCl 3rMeOH Ž3:1, vrv., Žl. the eluate with MeOH. MTT assay was performed on day 4. ŽA and B. The symbol B indicates medium only. Bars represent S.D. ŽC. Photomicrographs of macrophages cultured without Ža. or with Žb. 10 m grml of Fr. B in the presence of 20% L-cell supernatant for 4 days. Original magnification: =200 Žphase contrast microscopy..
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of cell membrane was often seen in cells incubated with Fr. B. These morphological changes were consistent with those of cells undergoing apoptosis w24x. Fraction B was further purified by silica gel chromatography. We obtained two fractions from the chromatography that were eluted by CHCl 3rMeOH Ž3:1. or by MeOH, respectively. As shown in Fig. 2B, the MeOH eluate almost completely abrogated MTT-reducing activity of macrophages, which was
consistent with the microscopic observation that the eluate induced macrophage cell death Žnot shown.. On the other hand, the CHCl 3rMeOH Ž3:1. eluate was not active, suggesting the active substances included in the extract have a relatively hydrophilic nature. Since the MeOH eluate contains saponin compounds, namely, securioside A and securioside B as active ingredients Žsee below., it was named ‘the securiosides preparation’ for simplicity, and in the
Fig. 3. ŽA. Time course of emergence of macrophages having apoptotic morphology. Starch-induced macrophages were cultured without Ž`. or with 1 m grml Žv ., 10 m grml Ž'. or 40 m grml ŽB. of the securiosides preparation in the presence of 20% L-cell-conditioned medium. The percentage of damaged cells on the indicated days was obtained by successively counting more than 200 cells using an inverted microscope. Bars represent S.D. ŽB. Analysis of DNA cleavage of cultured macrophages. Starch-induced macrophages were cultured with 40 m grml of the securiosides preparation in the presence of 20% L-cell supernatant for 4 days. The cells were stained by the TUNEL method and observed with phase contrast microscopy Ža., and DNA breaks represented by fluorescein were detected by fluorescence microscopy Žb..
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subsequent experiments, we characterized the activity present in S. inappendiculata using this preparation. 3.3. Reaction modes of securiosides We next examined the time-course of the emergence of apoptotic cells in macrophages to which M-CSF and the securiosides preparation had been added; the number of live or apoptotic cells, which could be easily discriminated by observation with phase-contrast microscopy, were differentially counted. As shown in Fig. 3A, the fraction at 1 and 10 m grml changed the morphology in more than half of the cells on day 2. After day 3, nearly all macrophages showed damaged morphology. On the other hand, 40 m grml of the sample caused a change in the great majority of cells on day 1. DNA cleavage and fragmentation are characteristics of apoptosis w24x. To confirm whether or not the macrophages cultured with the securiosides preparation underwent apoptosis, we checked whether the fraction induces cleavage in macrophage DNA by the TUNEL method. As shown in Fig. 3B, macrophages with a damaged shape caused by securiosides were TUNEL-positive, suggesting that they underwent an apoptotic cell death. To learn whether or not the substances in S. inappendiculata are effective only on macrophages stimulated with M-CSF, we examined the effect of
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the securiosides preparation on macrophages cultured without any growth factor and on GM-CSFstimulated macrophages. Unlike the result of macrophages cultured with M-CSF ŽFig. 4B., securiosides did not interfere with the MTT-reducing activity of untreated or GM-CSF-treated macrophages even at 10 m grml ŽFig. 4A and C.. These results suggest that active substances in S. inappendiculata selectively induced the apoptotic cell death of MCSF-stimulated macrophages. Target specificity of the active substances in S. inappendiculata was further explored by examining the effects of securiosides on tumor cell lines. The cytotoxic activity of the fraction was very weak against a macrophage-like cell line, J-774, even at 10 m grml ŽFig. 5A.; a similar tendency was seen in EL-4 and L-929 cells ŽFig. 5B and C.. Taken together, it is concluded that the effect of the substances in S. inappendiculata is very selective to peritoneal macrophages that are cultured with MCSF. 3.4. Cell death-inducing actiÕity of securioside A and securioside B As described elsewhere, we performed purification studies and determined the structures of two novel saponin compounds, namely, securioside A and securioside B w20x; their structures are depicted in Fig. 6. The cytotoxic activities of these com-
Fig. 4. Effect of securiosides on macrophages cultured with GM-CSF or macrophages cultured without any growth factor. Starch-induced macrophages were cultured with the indicated concentrations of the securiosides preparation without ŽA. or with 20% L-cell-conditioned medium ŽB. or recombinant mouse GM-CSF Ž100 ngrml. ŽC.. MTT assay was performed on day 4. Bars represent S.D.
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Fig. 5. Effect of securiosides on tumor cell lines. J.774.1 ŽA., EL-4 ŽB. or L-929 ŽC. were cultured with the indicated concentrations of the securiosides preparation. MTT assay was performed on day 4, respectively. Bars represent S.D.
pounds against macrophages or bone marrow cells cultured with L-cell supernatant are shown in Fig. 7A and B. They showed a cytotoxic effect on macrophages with almost the same dose–response relationship ŽED50 was between 0.1 and 1 m grml.. In contrast, their inhibitory concentrations against bone marrow cells stimulated with M-CSF were almost 10-fold higher than against macrophages. To learn further the specificity of the activity of securioside A and securioside B, we examined the effect on a normal cell type other than the cells in hematopoietic lineage. Fig. 7C showed the effect of securio-
sides on human dermal fibroblasts. As a result, the saponin compounds showed no inhibitory effect on human fibroblast up to 10 m grml. We next tried to elucidate whether the cell deathinducing activity against M-CSF-stimulated macrophages is common in saponin compounds. Securioside A and securioside B have a dimethoxycinnamoyl group which binds to L-fucose in each sugar chain ŽFig. 6.. To learn the importance of this acyl group in the cell death-inducing activity against macrophages, we prepared the deacylated forms of these compounds w20x, and their activities were tested.
Fig. 6. Structures of securioside A and securioside B.
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As shown in Fig. 7A, the activity was completely abrogated by deacylation, suggesting that the presence of a dimethoxycinnamoyl group is essential for the activity.
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Although the data is not shown, glycyrrhizin and soyasaponin did not cause macrophage death even at 100 m grml. Taken together, it is believed that the activity against M-CSF-stimulated macrophages is not common to all saponin compounds, but is specific to securioside A and securioside B, all having a dimethoxycinnamoyl group.
4. Discussion Macrophages play a central role in regulating many pathological conditions w1,2x. Not only the activation states of macrophages, but also local accumulation are important aspects in considering the regulatory effect of macrophages. However, relatively few trials had been performed to control the cell number of macrophages w25x. In the first step of this study, we found that a hot water extract of roots of S. inappendiculata contains cytotoxic compounds, which selectively act on macrophages, and that the saponin fraction selectively induced cell death of macrophages stimulated only with M-CSF-containing L-cell supernatant. As the purification studies proceeded, novel saponin compounds named securioside A and securioside B were obtained w20x. Cell death induction by these saponins was observed against macrophages cultured with recombinant mouse M-CSF Žnot shown., suggesting that M-CSF is the true active ingredient in L-cell supernatant which endows macrophages with their sensitivity to the toxicity of the saponins. The LPS contaminated in the partially purified securioside fraction and securiosides A and B did not affect the results, since in 1 m grml of these samples, LPS
Fig. 7. Cell death-inducing activity of securioside A and securioside B Žand their deacylated forms. on macrophages, bone marrow cells or human dermal fibroblasts. ŽA. Starch-induced macrophages were cultured with Ž`. securioside A, Ž^. securioside B, Žv . deacylated securioside A or Ž'. deacylated securioside B in the presence of 20% L-cell-conditioned medium. ŽB. Effects of securioside A and securioside B on M-CSF-stimulated bone marrow cells. Mouse bone marrow cells were cultured with Ž`. securioside A or Ž^. securioside B in the presence of 20% L-cell-conditioned medium. ŽC. Human dermal fibroblasts were cultured with Ž`. securioside A or Ž^. securioside B. MTT assay was performed on day 4 ŽA. or day 3 ŽB, C.. Bars represent S.D.
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carried with each sample into culture wells was estimated to be below 5 pgrml. It is worth noting that the saponins showed the toxic activity against M-CSF-stimulated macrophages, while they did not show the activity against GM-CSF-stimulated macrophages. Although studies for more extensively different types of cells are required to characterize the target specificity of the compounds, the macrophage-selective activity of the saponins was supported by the following evidences. Ž1. The partially purified saponin fraction did not affect three tumor cell lines including macrophagelike J774.1 cells up to 10 m grml. Ž2. All of them induced almost complete M-CSF-stimulated macrophage death at 1 m grml Ži.e., ; 0.7 m M., whereas they abrogated MTT reduction of bone marrow cells which were also stimulated with M-CSF at 10-fold higher concentration. Ž3. Securiosides did not inhibit growth of human dermal fibroblasts up to 10 m grml. Moreover, the purified saponins induced hemolysis against sheep red blood cells at more than 100 m grml Ždata not shown., suggesting that the toxic activity is not due to the detergent action that is characteristic to saponin compounds. It has been reported that some saponin compounds show growthinhibitory andror apoptosis-inducing activity against tumor cell lines w26–28x. However, to our knowledge, this is the first finding of materials that selectively induce macrophage death under the effect of M-CSF. The mechanism of cell death induction remains to be solved, although morphology and TUNEL analysis suggest that the death is caused by induction of apoptosis. One possibility is that the saponins from S. inappendiculata stimulated M-CSF-treated macrophages might produce cytotoxic cytokines, such as tumor necrosis factor ŽTNF. or Fas ligand in an autocrine or a paracrine fashion, because Fas–Fas ligand interactions reportedly induced apoptosis of human monocytes and macrophages w29x. Signal transduction of apoptosis induction initiated by Fas or TNF receptor stimulation reportedly requires activation of caspase 8, and another distinct pathway involves cytochrome c release from mitochondria and activation of caspase 9 w30,31x. Caspase 8 and caspase 9 are known to activate effector caspases such as caspase 3 w30x. We observed intracellular activation of caspase 3 and caspase 9, as well as
cytochrome c secretion from mitochondria in macrophages treated with securioside B and M-CSF, while activation of caspase 8 was not observed ŽYui, in preparation.. Since caspase 8 activation is located downstream of the TNF receptor or Fas stimulation, it is not likely that Fas or the TNF receptor-mediated pathway are involved in the cell death-inducing reaction of the saponins. There is growing evidence that macrophages internalize microbacterial products or putative endogenous materials such as several glycolipids, via surface receptors including CD1, CD14 and Toll-like receptors which sometimes recognize molecules having amphipathic property w32,33x, raising another possibility that specific receptorŽs. may emerge only on the surface of M-CSF-treated macrophages to bind and internalize the saponin compounds. In this respect, it is worth noting that the cell death-inducing activity against M-CSF-stimulated macrophages is not a common feature in these compounds: the deacylation of 3,4-dimethoxycinnamoyl group completely attenuated the activity of securioside A and securioside B, respectively, suggesting the importance of dimethoxycinnamoyl group in these saponins. In addition, we observed that 3,4-dimethoxycinnamoic acid, without or simultaneously with the deacylated saponin did not induce macrophage death at all Ždata not shown.. Together with the results that glycyrrhizin and soyasaponin did not show the activity, it is plausible that there are receptorŽs. on M-CSF-stimulated macrophages which recognize saponins having particular structural features, such as the presence of dimethoxycinnamoyl group. It is a future subject to elucidate how these active saponin compounds interact with cell surface molecules of macrophages. It has been observed that under many pathological conditions, local macrophages enter into a proliferating cycle w5–12x in which M-CSF may be an important factor inducing macrophage growth. Our current findings suggest that the saponin compounds from S. inappendiculata might be a novel approach to control the number of M-CSF-stimulated macrophages under such conditions. Since the compounds did not induce cell death of untreated macrophages, the basal functions of macrophages Žsuch as scavenging role, etc.. might not be severely influenced by treatment of the agents. Moreover, it may be advan-
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tageous in avoiding tissue destruction that the compounds less effectively affect the viability of cell types other than macrophages. Although more precise characterization regarding the target specificity of these compounds is required in the future study, securioside A and securioside B are believed to be leading compounds controlling the number of locally proliferating macrophages. They might also be new tools for use in analyzing the effect of M-CSF on macrophage function and growth.
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Acknowledgements The authors are grateful to Kaoru Yamada, Hiroko Kishi, Yukiko Iwao, Masaki Koido and Yuka Fukada for their technical assistance.
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experimental pancreatitis model. Arch Histol Cytol 1993; 56:75–82. Kerr PG, Nikolic-Paterson DJ, Lan HY, Tesch G, Rainone S, Atkins RC. Deoxyspergualin suppresses local macrophage proliferation in rat renal allograft rejection. Transplantation 1994;58:596–601. Lan HY, Nikolic-Paterson DJ, Atkins RC. Local macrophage proliferation in experimental Goodpasture’s syndrome. Nephrology 1995;1:151–6. Stanley ER, Chen DM, Lin HS. Induction of macrophage production and proliferation by a purified colony stimulating factor. Nature 1978;274:168–70. Chen BD-M, Clark CR, Chou TH. Granulocytermacrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor. Blood 1988;71: 997–1002. Chodakewits JA, Kupper TS, Coleman DL. Keratinocyte-derived granulocytermacrophage colony-stimulating factor induces DNA synthesis of peritoneal macrophages. J Immunol 1988;140:832–6. Yui S, Sasaki T, Miyazaki A, Horiuchi S, Yamazaki M. Induction of murine macrophage growth by modified LDLs. Arterioscler Thromb 1993;13:331–7. Yui S, Sasaki T, Araki N, Horiuchi S, Yamazaki M. Induction of macrophage growth by advanced end products of the Maillard reaction. J Immunol 1994;152:1943–9. Sasaki T, Horiuchi S, Yamazaki M, Yui S. Stimulation of macrophage DNA synthesis by polyanionic substances through binding to the macrophage scavenger receptor. Biol Pharm Bull 1996;19:449–55. Sakai M, Miyazaki A, Hakamata H, Kobori S, Shichiri M, et al. Endocytic uptake of lysophosphatidylcholine mediated by macrophage scavenger receptor plays a major role in oxidized low density lipoprotein-induced macrophage growth. J Atheroscler Thromb 1996;2:81–6. Kuroda M, Mimaki Y, Sashida Y, Kitahara M, Yamazaki M, et al. Securiosides A and B, novel acylated triterpene bisdesmosides with selective cytotoxic activity against M-CSFstimulated macrophages. Bioorg Med Chem Lett 2001;11: 371–4. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493–501. Yui S, Mikami M, Kitahara M, Yamazaki M. The inhibitory effect of lycorine on tumor cell apoptosis induced by polymorphonuclear leukocyte-derived calprotectin. Immunopharmacology 1998;40:151–62. Kerr JFR, Winterfold CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer 1994;73:2013– 26. Van Rooijen N, Sanders A. Elimination, blocking, and activation of macrophages: three of a kind? J Leukocyte Biol 1997;62:702–9.
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w26x Mimaki Y, Sashida Y, Kuroda M, Nishino A, Satomi Y, Nisino H. Inhibitory effects of steroidal saponins on 12-O-tetradecanoylphorbol-13-acetate ŽTPA.-enhanced 32 P-incorporation into phospholipids of Hela cells and proliferation of human malignant tumor cells. Biol Pharm Bull 1995;18:467– 9. w27x Hirano T, Oka K, Mimaki Y, Kuroda M, Sashida Y. Potent growth Inhibitory activity of a novel Ornithogalum cholestane glycoside on human cells: induction of apoptosis in promyelocytic leukemia HL-60 cells. Life Sci 1996;58:789–98. w28x Lee SJ, Ko WG, Kim JH, Sung JH, Moon CK, Lee BH. Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of caspase-3 protease. Biochem Pharmacol 2000;60:677–85.
w29x Kiener PA, Davis PM, Starling GC, Mehlin C, Klebanoff SJ, et al. Differential induction of apoptosis by Fas–Fas ligand interactions in human monocytes and macrophages. J Exp Med 1997;185:1511–6. w30x Strasser A, O’Connor L, Dixit VM. Apoptosis signalling. Annu Rev Biochem 2000;69:217–45. w31x Boldin MP, Goncharov TM, Golsev YV, Wallach D. Involvement of MACH, a novel MORT1rFADD-interacting protease, in FasrAPO- and TNF receptor-induced cell death. Cell 1996;85:803–15. w32x Park S-H, Bendelac A. CD 1-restricted T-cell responses and microbial infection. Nature 2000;406:788–91. w33x Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406:782–7.
Macrophage-oriented cytotoxic activity of novel triterpene saponins extracted from roots of Securidaca inappendiculata Satoru Yui a,) , Kazuyoshi Ubukata a , Kazumi Hodono a , Mikio Kitahara b, Yoshihiro Mimaki c , Minpei Kuroda c , Yutaka Sashida c , Masatoshi Yamazaki a a
Faculty of Pharmaceutical Sciences, Teikyo UniÕersity, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan Takasago Research Laboratories, Research Institute, Kaneka Corporation, Miyamae-cho, Takasago-cho, Takasago, Hyogo 676-8688, Japan Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo UniÕersity of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan b
c
Received 1 March 2001; received in revised form 31 May 2001; accepted 27 June 2001
Abstract It is recognized that macrophages in peripheral tissues often proliferate under pathological conditions such as tumors, inflammation and atherosclerosis. Because the growth state of macrophages is believed to be a factor regulating the pathological process of the diseases, substances that regulate macrophage growth or survival may be useful for disease control. In this paper, we identified the activity inhibiting macrophage growth in a hot water extract of roots of Securidaca inappendiculata. The extract markedly inhibited macrophage colony-stimulating factor ŽM-CSFrCSF-1.-induced growth of macrophages, whereas it exerted a less potent effect on growth of Concanavalin A ŽCon A.-stimulated thymocytes or M-CSF-stimulated bone marrow cells. The inhibition of macrophage growth was caused by a cytotoxic effect rather than a cytostatic effect. Cell death was due to the induction of apoptosis, as judged by staining with terminal deoxynucleotidyl transferase-mediated d-UTP nick end labelling ŽTUNEL.. The cytotoxic activity seemed to be specific to peripheral macrophages; it showed a weak effect on the growth and survival of tumor cell lines including a macrophage-like cell line, J-774.1. Moreover, the saponin fraction induced apoptotic cell death of macrophages only when they were stimulated by M-CSF; it did not affect the viability of macrophages cultured without M-CSF or with granulocytermacrophage-CSF. We determined the structures of the two active triterpene saponin compounds in the fraction, named securioside A and securioside B having a 3,4-dimethoxycinnamic group which is essential for the cell death-inducing activity. They are believed to be the primary compounds of new drugs for the treatment of pathological states in which macrophage proliferation occurs. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Macrophages; Apoptosis; M-CSF; Saponin compounds
1. Introduction
)
Corresponding author. Tel.: q81-426-85-3736; fax: q81-42685-2574. E-mail address: [email protected] ŽS. Yui..
It is widely recognized that macrophages play a central role in the regulation of inflammatory and immunological reactions through various functions including phagocytic elimination of foreign or dena-
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 2 6 - 6
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tured substances, and secretion of various cytokines or reactive oxygen intermediates w1,2x. Under local pathological conditions, the function of macrophages depends on qualitative and quantitative aspects, namely, their activation state and their accumulation in local sites. There are cumulative observations of the peripheral proliferation of macrophages in pathological tissues such as tumors w3,4x, atherosclerotic lesions w5–8x and several inflammatory sites w9–12x. The protein factors, macrophage colony-stimulating factor ŽM-CSFrCSF-1. and granulocytermacrophage colony-stimulating factor ŽGM-CSF. are growth factors for peripheral macrophages w13–15x. In addition to them, we reported that materials to be scavenged by macrophages via scavenger receptorŽs. induce macrophage growth in the mediation of GMCSF w16–18x. The normally undividing macrophages may respond to the above factors and enter a cell division cycle under pathological conditions. The local proliferation is thought to contribute to macrophage accumulation, and it may also influence the pathological process. For example, in atherosclerotic lesions, the proliferation of lipid-laden macrophages that scavenge oxidized low density lipoprotein ŽLDL. may be implicated in the accumulation of cholesteryl esters in the intima of blood vessels, as well as in the formation of atherosclerotic plaques w5–7,16,19x. Under many other inflammatory conditions, macrophage expansion in situ is believed to influence the consequence of a disease w9–12x since, as described above, macrophage is located in a central position of inflammation regulation. Accordingly, how to inhibit macrophage proliferation andror eliminate macrophages in the mitotic stage is an important aspect of the regulation of inflammatory diseases. To identify a substance that inhibits macrophage proliferation, we performed a screening study among hot water extracts of plant materials that have been traditionally used as Chinese medicines and are known to have anti-inflammatory activity. Among these materials, we found that the extract of the roots of Securidaca inappendiculata contains saponin compounds that induce cytotoxic activity against MCSF-stimulated macrophages. We identified novel triterpene saponins as cytotoxic molecules against macrophages and they were named securioside A and securiosides B w20x. In this report, we further
extended the characterization of the cytotoxic activity of these saponins against macrophages.
2. Materials and methods 2.1. Reagents Soyasaponin and glycyrrhizin were purchased from Wako ŽPure Chemicals, Osaka, Japan.. Concanavalin A ŽCon A. was from E.Y. Laboratories ŽSan Mateo, CA.. Recombinant mouse M-CSF and GM-CSF were purchased from Genzyme ŽCambridge, MA.. 2.2. Mice Male C3HrHe mice and C3HrHeJ mice were purchased from breeding colonies in Japan SLC ŽShizuoka, Japan.. 2.3. Macrophage culture Peritoneal cells were obtained 3–5 days after i.p. injection of 30 mg of starch into each C3HrHe mouse. These cells were suspended in RPMI 1640 medium ŽNissui Seiyaku, Tokyo, Japan. supplemented with 5% heated-inactivated fetal calf serum ŽFCS, Summit, Ft. Collins, CO, USA., penicillin Ž100 Urml. and Kanamycin Ž60 m grml.. For wmethyl3 Hx-thymidine Žw3 Hx-TdR. incorporation assay and MTT assay, the peritoneal cells were incubated in 96-well microplates ŽCorning, Corning, NY. at 2 = 10 4 cellsrwell. For morphological observation, these cells were incubated in 24-well microplates ŽNunc, OK-4000, Roskilde, Denmark. at 0.5 = 10 5 cellsrwell. The cells were incubated for 90 min at 37 8C in a CO 2-incubator to allow them to adhere to the culture plates. The medium was then removed, and the nonadherent cells were removed by three vigorous washings with prewarmed PBS solution. More than 95% of the adherent cells were judged to be macrophage both by Giemsa staining and carbon particle uptake. These macrophages were added with the sample to be tested and simultaneously with L-cell-conditioned medium that was used as crude M-CSF w13x. The cells were cultured at 37 8C in a humidified atmosphere of 5% CO 2 in air
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with 0.2 ml Žfor 96 well plates. or 1 ml Žfor 24 well plates. of medium without changing the medium. 2.4. [3H]-TdR incorporation The incorporation of 3 H-TdR into cultured macrophages was measured as described w18x. Briefly, after the indicated culture periods, w3 Hx-TdR Ž2960 TBqr mmol, New England Nuclear, Boston, MA. was added to each plate at 37 kBqrml and incubated for 18 h. The medium was discarded, and the cells were dissolved in 100 m l of 0.5% sodium dodecyl sulfate, followed by addition of 100 m l of cold 10% trichloroacetic acid. The resulting trichloroacetic acidinsoluble material was collected on filters with Labo mash LM-101 ŽFutaba Medical, Tokyo.. The filters were dried, and their radioactivity was counted in a liquid scintillation spectrophotometer. All experiments were performed in triplicate. 2.5. MTT assay Macrophage cell death was evaluated by MTT assay w21x. Macrophages in 96 well plates were added with 25 m lrwell of 3-Ž4,5-dimetyl-2-thiazolyl.-2,5-diphenyl-2 H-tetrazolium bromide ŽMTT, 5 mgrml. and plates were incubated for an additional 3 h. Then, 150 m l of the supernatants was discarded, 100 m l of acid-isopropanol solution Ž0.04 N HCl in 2-propanol. was added to each well and the optical density Ž588 nm. was measured with a microplate reader ŽMTP-100, Corona Electric, Ibaragi, Japan.. Experiments were performed with duplicate determinations and results were expressed as relative values Ž% of control. in which each mean value was compared with that of medium alone. 2.6. Microscopic obserÕation Morphological observations of cultured macrophages were made by inverted, phase-contrast microscopy. DNA breaks in cultured macrophages were detected in situ by the terminal deoxynucleotidyl transferase ŽTdT.-mediated dUTP nick end labeling ŽTUNEL. method, which was originally described by Gavrieli et al. w22x, using a kit ŽIn Situ Cell Death Detection Kit, fluorescein, Boeringer-Mannheim,
1991
Mannheim, FRG.. The TUNEL reaction enzymatically labels free 3X-OH termini of DNA strand breaks with fluorescein-dUTP with TdT. The fluorescein incorporated into cells was observed by fluorescence microscopy. 2.7. Other cells Bone marrow cells were collected from femurs of C3HrHe mice, and cultured in the presence of 10% L-cell-conditioned medium. 3 H-TdR was pulsed on day 3 for 16 h and cells were harvested as described in macrophage culture. Thymocytes were obtained from C3HrHeJ mice. The cells Ž1.5 = 10 6 cellsr200 m l. were cultured in wells of 96-well plates with 5 m grml of Con A. 3 H-TdR was pulsed on day 2 for 5 h and cells were immediately trapped in glass filters using water as a washing solution. The mouse fibrosarcoma line, L-929, was maintained in MEM supplemented with 5% calf serum. The macrophagelike cell line, J774.1 obtained from HSRRB Cell Bank ŽOsaka, Japan. and EL-4 mouse thymoma were maintained in RPMI 1640 medium supplemented with 5% FCS. Human dermal fibroblasts were purchased from Cell Systems ŽKirkland, WA. and were maintained in RPMI 1640 medium supplemented with 10% FCS. These tumor cell lines and human dermal fibroblasts were cultured in wells of 96-well plates at the concentration of 1 = 10 4 cellsrwell, respectively. 2.8. Plant materials The plant materials Ž59 species. used for screening study were from the sources listed elsewhere, in which we had screened the inhibitory substance for calprotectin-induced apoptosis w23x. 2.9. Purification of the substances inducing macrophage cell death The dried roots of S. inappendiculata Hassk. Ž154 g. were extracted with distilled water Žtotal 3000 ml. at 110 8C for 3 h. After the residue was removed by centrifugation, the supernatant was lyophilized, and 8.85-g dried powder was obtained. A part of the dried extract was dissolved in water and was partitioned into aqueous phase and the ether fraction
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using extraction with diethyl ether. The aqueous phase was thereafter extracted with butanol to produce the butanol fraction and the resultant aqueous fraction. Each fraction was evaporated and dissolved in MeOH. In a pilot experiment, 120 mg of hot water extract yielded 16 mg of ether fraction, 24 mg of butanol fraction and 47 mg of aqueous fraction, respectively. The butanol fraction, termed Fr. B Ž500 mg from an experiment on a larger scale. was further purified with Wako-gel C-200 column chromatography ŽWako.. After elution of CHCl 3rMeOH Ž3:1, vrv., which resulted in a 148-mg residue after evaporation, the fraction including cytotoxic activity against macrophages was eluted with MeOH. Two purified saponins, namely, securioside A and securioside B and their deacylated forms were prepared from the MeOH eluate as described elsewhere w20x. The LPS content of the MeOH eluate fraction, securioside A and securioside B was measured by Toxicolor ŽSeikagaku, Tokyo, Japan., and was estimated to be 2.0, 3.4 and 2.5 ngrmg, respectively.
3. Results 3.1. Screening of hot water extracts of plant materials to identify substances inhibiting macrophage growth To find substances having inhibitory activity for macrophage growth, we screened various hot water extracts of 59 plant species that have been used as Chinese medicine w23x. The extracts were added to macrophage cultures in the presence of L-cell-conditioned medium as a macrophage growth factor. Three
Fig. 1. Inhibitory activities of the hot water extract from roots of S. inappendiculata against 3 H-TdR incorporation of macrophages, thymocytes and bone marrow cells. ŽA. Starch-induced macrophages were cultured with the indicated concentrations of hot water extract in the presence of 20% L-cell-conditioned medium for 6 days. ŽB. Thymocytes were cultured with the indicated concentrations of hot water extract in the presence of Con A for 2 days. ŽC. Bone marrow cells were cultured with the indicated concentrations of hot water extract in the presence of 10% L-cell-conditioned medium for 3 days. 3 H-TdR incorporation into these cells was measured as described in Materials and Methods. Bars represent S.D.
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of these extracts Ž S. inappendiculata, Cimicifuga foetida and Arctium lappa. showed strong inhibitory activity Ždata not shown.. In this paper, we studied the properties of the substances present in S. inappendiculata. Fig. 1A depicts the dose–response relationship of the inhibitory effect of the crude extract of S. inappendiculata on macrophage 3 H-TdR incorporation; the inhibition was seen from 0.63 m grml. To access the specificity of the activity of this extract, we examined its effects on bone marrow cells and thymocytes. As shown in Fig. 1B and C, only a high concentration of the extract Ž40 m grml. reduced 3 H-TdR incorporation into thymocytes or bone marrow cells which were stimulated with Con A or M-CSF, respectively, suggesting that macrophages are specifically sensitive to the substanceŽs. included in S. inappendiculata.
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3.2. Partial purification of the actiÕe substances To purify the active substances, we performed fractionation of the extract by sequential extraction using diethyl ether and then butanol. As shown in Fig. 2A, the butanol extract ŽFr. B. showed higher specific activity than the crude extract, while the aqueous fraction and ether fraction showed weaker activity than the crude extract. To ascertain whether the inhibition is due to the cytostatic effect or the cytocidal effect, the morphology of macrophages cultured without or with Fr. B is shown in Fig. 2C—a and b, respectively. More than half of the cells cultured with Fr. B for 2 days showed small, shrunken morphology. Although some damaged cells still adhered to the culture substrate, they were no longer spreading. The breb formation
Fig. 2. Macrophage growth-inhibiting or toxic activity of partially purified samples partitioned from the extract of S. inappendiculata. ŽA. Starch-induced macrophages were cultured with the following samples obtained by organic solvent extraction of the hot water extract in the presence of 20% L-cell-conditioned medium. Žv . Crude hot water extract, Ž`. ether-extracted fraction, Ž^. butanol fraction ŽFr. B., ŽI. aqueous fraction. 3 H-TdR incorporation into macrophages was assayed on day 6. ŽB. Starch-induced macrophages were cultured with the samples obtained by silicic column chromatography of Fr. B in the presence of 20% L-cell-conditioned medium: Ž^. Fr. B, Že. the eluate with CHCl 3rMeOH Ž3:1, vrv., Žl. the eluate with MeOH. MTT assay was performed on day 4. ŽA and B. The symbol B indicates medium only. Bars represent S.D. ŽC. Photomicrographs of macrophages cultured without Ža. or with Žb. 10 m grml of Fr. B in the presence of 20% L-cell supernatant for 4 days. Original magnification: =200 Žphase contrast microscopy..
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of cell membrane was often seen in cells incubated with Fr. B. These morphological changes were consistent with those of cells undergoing apoptosis w24x. Fraction B was further purified by silica gel chromatography. We obtained two fractions from the chromatography that were eluted by CHCl 3rMeOH Ž3:1. or by MeOH, respectively. As shown in Fig. 2B, the MeOH eluate almost completely abrogated MTT-reducing activity of macrophages, which was
consistent with the microscopic observation that the eluate induced macrophage cell death Žnot shown.. On the other hand, the CHCl 3rMeOH Ž3:1. eluate was not active, suggesting the active substances included in the extract have a relatively hydrophilic nature. Since the MeOH eluate contains saponin compounds, namely, securioside A and securioside B as active ingredients Žsee below., it was named ‘the securiosides preparation’ for simplicity, and in the
Fig. 3. ŽA. Time course of emergence of macrophages having apoptotic morphology. Starch-induced macrophages were cultured without Ž`. or with 1 m grml Žv ., 10 m grml Ž'. or 40 m grml ŽB. of the securiosides preparation in the presence of 20% L-cell-conditioned medium. The percentage of damaged cells on the indicated days was obtained by successively counting more than 200 cells using an inverted microscope. Bars represent S.D. ŽB. Analysis of DNA cleavage of cultured macrophages. Starch-induced macrophages were cultured with 40 m grml of the securiosides preparation in the presence of 20% L-cell supernatant for 4 days. The cells were stained by the TUNEL method and observed with phase contrast microscopy Ža., and DNA breaks represented by fluorescein were detected by fluorescence microscopy Žb..
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subsequent experiments, we characterized the activity present in S. inappendiculata using this preparation. 3.3. Reaction modes of securiosides We next examined the time-course of the emergence of apoptotic cells in macrophages to which M-CSF and the securiosides preparation had been added; the number of live or apoptotic cells, which could be easily discriminated by observation with phase-contrast microscopy, were differentially counted. As shown in Fig. 3A, the fraction at 1 and 10 m grml changed the morphology in more than half of the cells on day 2. After day 3, nearly all macrophages showed damaged morphology. On the other hand, 40 m grml of the sample caused a change in the great majority of cells on day 1. DNA cleavage and fragmentation are characteristics of apoptosis w24x. To confirm whether or not the macrophages cultured with the securiosides preparation underwent apoptosis, we checked whether the fraction induces cleavage in macrophage DNA by the TUNEL method. As shown in Fig. 3B, macrophages with a damaged shape caused by securiosides were TUNEL-positive, suggesting that they underwent an apoptotic cell death. To learn whether or not the substances in S. inappendiculata are effective only on macrophages stimulated with M-CSF, we examined the effect of
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the securiosides preparation on macrophages cultured without any growth factor and on GM-CSFstimulated macrophages. Unlike the result of macrophages cultured with M-CSF ŽFig. 4B., securiosides did not interfere with the MTT-reducing activity of untreated or GM-CSF-treated macrophages even at 10 m grml ŽFig. 4A and C.. These results suggest that active substances in S. inappendiculata selectively induced the apoptotic cell death of MCSF-stimulated macrophages. Target specificity of the active substances in S. inappendiculata was further explored by examining the effects of securiosides on tumor cell lines. The cytotoxic activity of the fraction was very weak against a macrophage-like cell line, J-774, even at 10 m grml ŽFig. 5A.; a similar tendency was seen in EL-4 and L-929 cells ŽFig. 5B and C.. Taken together, it is concluded that the effect of the substances in S. inappendiculata is very selective to peritoneal macrophages that are cultured with MCSF. 3.4. Cell death-inducing actiÕity of securioside A and securioside B As described elsewhere, we performed purification studies and determined the structures of two novel saponin compounds, namely, securioside A and securioside B w20x; their structures are depicted in Fig. 6. The cytotoxic activities of these com-
Fig. 4. Effect of securiosides on macrophages cultured with GM-CSF or macrophages cultured without any growth factor. Starch-induced macrophages were cultured with the indicated concentrations of the securiosides preparation without ŽA. or with 20% L-cell-conditioned medium ŽB. or recombinant mouse GM-CSF Ž100 ngrml. ŽC.. MTT assay was performed on day 4. Bars represent S.D.
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Fig. 5. Effect of securiosides on tumor cell lines. J.774.1 ŽA., EL-4 ŽB. or L-929 ŽC. were cultured with the indicated concentrations of the securiosides preparation. MTT assay was performed on day 4, respectively. Bars represent S.D.
pounds against macrophages or bone marrow cells cultured with L-cell supernatant are shown in Fig. 7A and B. They showed a cytotoxic effect on macrophages with almost the same dose–response relationship ŽED50 was between 0.1 and 1 m grml.. In contrast, their inhibitory concentrations against bone marrow cells stimulated with M-CSF were almost 10-fold higher than against macrophages. To learn further the specificity of the activity of securioside A and securioside B, we examined the effect on a normal cell type other than the cells in hematopoietic lineage. Fig. 7C showed the effect of securio-
sides on human dermal fibroblasts. As a result, the saponin compounds showed no inhibitory effect on human fibroblast up to 10 m grml. We next tried to elucidate whether the cell deathinducing activity against M-CSF-stimulated macrophages is common in saponin compounds. Securioside A and securioside B have a dimethoxycinnamoyl group which binds to L-fucose in each sugar chain ŽFig. 6.. To learn the importance of this acyl group in the cell death-inducing activity against macrophages, we prepared the deacylated forms of these compounds w20x, and their activities were tested.
Fig. 6. Structures of securioside A and securioside B.
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As shown in Fig. 7A, the activity was completely abrogated by deacylation, suggesting that the presence of a dimethoxycinnamoyl group is essential for the activity.
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Although the data is not shown, glycyrrhizin and soyasaponin did not cause macrophage death even at 100 m grml. Taken together, it is believed that the activity against M-CSF-stimulated macrophages is not common to all saponin compounds, but is specific to securioside A and securioside B, all having a dimethoxycinnamoyl group.
4. Discussion Macrophages play a central role in regulating many pathological conditions w1,2x. Not only the activation states of macrophages, but also local accumulation are important aspects in considering the regulatory effect of macrophages. However, relatively few trials had been performed to control the cell number of macrophages w25x. In the first step of this study, we found that a hot water extract of roots of S. inappendiculata contains cytotoxic compounds, which selectively act on macrophages, and that the saponin fraction selectively induced cell death of macrophages stimulated only with M-CSF-containing L-cell supernatant. As the purification studies proceeded, novel saponin compounds named securioside A and securioside B were obtained w20x. Cell death induction by these saponins was observed against macrophages cultured with recombinant mouse M-CSF Žnot shown., suggesting that M-CSF is the true active ingredient in L-cell supernatant which endows macrophages with their sensitivity to the toxicity of the saponins. The LPS contaminated in the partially purified securioside fraction and securiosides A and B did not affect the results, since in 1 m grml of these samples, LPS
Fig. 7. Cell death-inducing activity of securioside A and securioside B Žand their deacylated forms. on macrophages, bone marrow cells or human dermal fibroblasts. ŽA. Starch-induced macrophages were cultured with Ž`. securioside A, Ž^. securioside B, Žv . deacylated securioside A or Ž'. deacylated securioside B in the presence of 20% L-cell-conditioned medium. ŽB. Effects of securioside A and securioside B on M-CSF-stimulated bone marrow cells. Mouse bone marrow cells were cultured with Ž`. securioside A or Ž^. securioside B in the presence of 20% L-cell-conditioned medium. ŽC. Human dermal fibroblasts were cultured with Ž`. securioside A or Ž^. securioside B. MTT assay was performed on day 4 ŽA. or day 3 ŽB, C.. Bars represent S.D.
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carried with each sample into culture wells was estimated to be below 5 pgrml. It is worth noting that the saponins showed the toxic activity against M-CSF-stimulated macrophages, while they did not show the activity against GM-CSF-stimulated macrophages. Although studies for more extensively different types of cells are required to characterize the target specificity of the compounds, the macrophage-selective activity of the saponins was supported by the following evidences. Ž1. The partially purified saponin fraction did not affect three tumor cell lines including macrophagelike J774.1 cells up to 10 m grml. Ž2. All of them induced almost complete M-CSF-stimulated macrophage death at 1 m grml Ži.e., ; 0.7 m M., whereas they abrogated MTT reduction of bone marrow cells which were also stimulated with M-CSF at 10-fold higher concentration. Ž3. Securiosides did not inhibit growth of human dermal fibroblasts up to 10 m grml. Moreover, the purified saponins induced hemolysis against sheep red blood cells at more than 100 m grml Ždata not shown., suggesting that the toxic activity is not due to the detergent action that is characteristic to saponin compounds. It has been reported that some saponin compounds show growthinhibitory andror apoptosis-inducing activity against tumor cell lines w26–28x. However, to our knowledge, this is the first finding of materials that selectively induce macrophage death under the effect of M-CSF. The mechanism of cell death induction remains to be solved, although morphology and TUNEL analysis suggest that the death is caused by induction of apoptosis. One possibility is that the saponins from S. inappendiculata stimulated M-CSF-treated macrophages might produce cytotoxic cytokines, such as tumor necrosis factor ŽTNF. or Fas ligand in an autocrine or a paracrine fashion, because Fas–Fas ligand interactions reportedly induced apoptosis of human monocytes and macrophages w29x. Signal transduction of apoptosis induction initiated by Fas or TNF receptor stimulation reportedly requires activation of caspase 8, and another distinct pathway involves cytochrome c release from mitochondria and activation of caspase 9 w30,31x. Caspase 8 and caspase 9 are known to activate effector caspases such as caspase 3 w30x. We observed intracellular activation of caspase 3 and caspase 9, as well as
cytochrome c secretion from mitochondria in macrophages treated with securioside B and M-CSF, while activation of caspase 8 was not observed ŽYui, in preparation.. Since caspase 8 activation is located downstream of the TNF receptor or Fas stimulation, it is not likely that Fas or the TNF receptor-mediated pathway are involved in the cell death-inducing reaction of the saponins. There is growing evidence that macrophages internalize microbacterial products or putative endogenous materials such as several glycolipids, via surface receptors including CD1, CD14 and Toll-like receptors which sometimes recognize molecules having amphipathic property w32,33x, raising another possibility that specific receptorŽs. may emerge only on the surface of M-CSF-treated macrophages to bind and internalize the saponin compounds. In this respect, it is worth noting that the cell death-inducing activity against M-CSF-stimulated macrophages is not a common feature in these compounds: the deacylation of 3,4-dimethoxycinnamoyl group completely attenuated the activity of securioside A and securioside B, respectively, suggesting the importance of dimethoxycinnamoyl group in these saponins. In addition, we observed that 3,4-dimethoxycinnamoic acid, without or simultaneously with the deacylated saponin did not induce macrophage death at all Ždata not shown.. Together with the results that glycyrrhizin and soyasaponin did not show the activity, it is plausible that there are receptorŽs. on M-CSF-stimulated macrophages which recognize saponins having particular structural features, such as the presence of dimethoxycinnamoyl group. It is a future subject to elucidate how these active saponin compounds interact with cell surface molecules of macrophages. It has been observed that under many pathological conditions, local macrophages enter into a proliferating cycle w5–12x in which M-CSF may be an important factor inducing macrophage growth. Our current findings suggest that the saponin compounds from S. inappendiculata might be a novel approach to control the number of M-CSF-stimulated macrophages under such conditions. Since the compounds did not induce cell death of untreated macrophages, the basal functions of macrophages Žsuch as scavenging role, etc.. might not be severely influenced by treatment of the agents. Moreover, it may be advan-
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tageous in avoiding tissue destruction that the compounds less effectively affect the viability of cell types other than macrophages. Although more precise characterization regarding the target specificity of these compounds is required in the future study, securioside A and securioside B are believed to be leading compounds controlling the number of locally proliferating macrophages. They might also be new tools for use in analyzing the effect of M-CSF on macrophage function and growth.
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Acknowledgements The authors are grateful to Kaoru Yamada, Hiroko Kishi, Yukiko Iwao, Masaki Koido and Yuka Fukada for their technical assistance.
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