Capillin, a major constituent of Artemisia capillaris Thunb. flower essential oil, induces apoptosis through the mitochondrial pathway in human leukemia HL-60 cells.
Background: Natural products are one of the most important sources of drugs used in pharmaceutical therapeutics. Screening of several natural products in the search for novel anticancer agents against human leukemia HL-60 cells led us to identify potent apoptosis-inducing activity in the essential oil fraction from Artemisia capillaris Thunb. flower.
Methods: The cytotoxic effects of extracts were assessed on human leukemia HL-60 cells by XTT assay. Induction of apoptosis was assessed by analysis of DNA fragmentation and nuclear morphological change. The plant name was checked with the plant list website (http://www.theplantlist.org).
Results: A purified compound from the essential oil fraction from Artemisia capillaris Thunb. flower that potently inhibited cell growth in human leukemia HL-60 cells was identified as capillin. The cytotoxic effect of capillin in cells was associated with apoptosis. When HL-60 cells were treated with 10-6 M capillin for 6 h, characteristic features of apoptosis such as DNA fragmentation and nuclear fragmentation were observed. Moreover, activation of c-Jun N-terminal kinase (JNK) was detected after treatment with capillin preceding the appearance of characteristic properties of apoptosis. Release of cytochrome c from mitochondria was also observed in HL-60 cells that had been treated with capillin.
Conclusion: Capillin induces apoptosis in HL-60 cells via the mitochondrial apoptotic pathway, which might be controlled through JNK signaling. Our results indicate that capillin may be a potentially useful anticancer drug that could enhance therapeutic efficacy.
Artemisia capillaris Thunb.
Capillin (PubChem CID: 10321)
Although cancer cells generally do not differentiate or undergo apoptosis, they can be induced to differentiate and undergo apoptosis by certain chemical agents. Various chemotherapeutic agents that are used to treat cancers, such as adriamycin (Friesen et al. 1996), cisplatin (Barry et al. 1990; Kaufmann 1989), and taxol (Bhalla et al. 1993) have been reported to have apoptosis-inducing activity. Therefore, the induction of apoptosis in cancer cells has become an indicator of the effect of anticancer drugs, and chemical agents with strong apoptosis-inducing activity are expected to have potential for use as anticancer drugs. We have identified a variety of inducers of differentiation and apoptosis in cancer cells, which include isoprenoid compounds such as geranylgeranylacetone (Sakai et al. 1993), geranylgeraniol (Ohizumi et al. 1995), and vitamin K2 (Sakai et al. 1994; Sibayama-Imazu et al. 2008), as well as inhibitors of topoisomerase such as camptothecin (Chou et al. 1990) and etoposide (Jing et al. 1994),
Apoptosis is regulated by a variety of signaling pathways, including members of the mitogen-activated protein kinase (MAPK) superfamily. JNK/SAPK and p38, also known as a stress-activated protein kinase, are activated by several extracellular stimuli, such as UV light, TNF-[alpha], and osmotic shock (Derijard et al. 1994; Galcheva-Gargova et al. 1994; Sluss et al. 1994). As apoptosis is induced by a variety of extracellular stresses and signals, involvement of stress-activated protein kinases has been proposed in apoptotic processes that are induced in differentiated PCI 2 cells by NGF deprivation (Xia et al. 1995) or in U937 cells by ceramide treatment (Verheij et al. 1996). In the case of apoptosis induced by the topoisomerase inhibitor [beta]-lapachone in HL-60 cells, only JNK and not p38 was activated, and subsequent activation of caspase 3 was observed (Shiah et al. 1999). In the present study, we investigated intracellular signaling pathways that lead to induction of apoptosis in response to essential oils from Artemisia capillaris Thunb. flower in human leukemia HL-60 cells.
Artemisia species have been used as food additives and traditional herbal medicines, particularly in treating diseases such as cancer, inflammation, malaria, hepatitis, and microbial infections (Aniya et al. 2000; Gilani et al. 2005; Kordali et al. 2005). Of these species, the flowers of Artemisia capillaris Thunb., which have been used as an important crude drug (Japanese name "inchinko") since antiquity, exhibit a wide variety of pharmacological activities including anti-inflammatory, antiallergic, and anticancer activities (Chen et al. 1994; Lee et al. 2003). Recently, there has been an increasing interest in the use of essential oils as medical agents, because essential oils have been reported to have chemopreventive potential via induction of apoptosis in several tumor cell lines (Buhagiar et al. 1999; Paik et al. 2005). These observations suggest that essential oils of Artemisia capillaris Thunb. flower might be beneficial apoptosis inducers in cancer cells. We found, in the present study, that, among various compounds in the essential oils of Artemisia capillaris Thunb. flower, capillin had the strongest apoptosis-inducing activity with HL-60 cells. Capillin is a constituent of essential oil from some Artemisia species, and displays important biological activities, such as antitumor, antibacterial, antimicrobial, and antifungal activities (Dembitsky 2006; Joshi et al. 2010; Sharopov and Setzer 2011). We also demonstrated that capillin strongly induced JNK/SAPK activity without activation of other members of the MAPK family, such as ERK. The characteristics of apoptosis induced by capillin in HL-60 cells are described in the present paper.
Materials and methods
Reagents, cell lines, and cell culture
VP16 and XTT were purchased from Sigma. Phospho-SAPK/JNK (Thr183/Tyr185) mouse monoclonal antibody, SAPK/JNK (56G8) rabbit monoclonal antibody, phosphor-p44/42 MAPK (Thr202/Tyr204) antibody, and p44/42 MAPK antibody were obtained from Cell Signaling Technology. A monoclonal antibody against cytochrome c (7H8.2C12) was purchased from BD Pharmingen. Human leukemia HL-60 cells were provided by the Japanese Cancer Resource Bank. The flower of Artemisia capillaris Thunb. was purchased from Uchida Wakanyaku Ltd. (Lot. No. 9B9004). HL-60 cells were maintained in RPMI 1640 medium (Invitrogen), supplemented with 10% heatin-activated fetal calf serum, in an atmosphere of 5% C[O.sub.2] at 37[degrees]C. The cells were seeded at a concentration of 2-3 x 105 cells/ml, and logarithmic growth was maintained by passing them every 2-3 days. WI-38 normal human fibroblasts, obtained from the American Type Culture Collection, were maintained in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum under the same growth conditions.
Isolation of capillin and capillene
Commercial Artemisia capillaris Thunb. flowers (100 g) were placed in a 11 hard glass stoppered flask with 500 ml of water. After setting up an apparatus for essential oil determination with a reflux condenser, the contents of the flask were heated between 130[degrees]C and 150[degrees]C to boil. The essential oil was captured in xylene (2 ml), as described in the section of the essential oil contents of the Japanese Pharmacopoeia. After evaporation of the xylene, the essential oil (300 mg) was applied to an HPLC [ODS, C[H.sub.3]CN-[H.sub.2]O (17:3)] to give capillin (1-phenylhexa-2,4-diyn-1-one) (Rt: 8.0 min, 85 mg) and capillene (hexa-2,4-diyn-1-ylbenzene) (Rt: 8.6 min, 21 mg), respectively.
Isolation of capillarisin and 6,7-dimethylesculetin
Commercial Artemisia capillaris Thunb. flowers (100 g) were extracted with boiling water (1.8 L) for 1 h, and the aqueous extract was applied to an Amberlite XAD-II column ([H.sub.2]O [right arrow] MeOH) to give a MeOH eluate fraction (6.5 g). The MeOH eluate fraction was subjected to Sephadex LH-20 (MeOH) chromatography to give three fractions (B-1 to B-3). Fraction B-2 (2.5 g) was subjected to repeated silica gel column chromatography [CH[Cl.sub.3]-MeOH-[H.sub.2]O (19:1), n-hexane-EtOAc (7:3)] to give capillarisin (5,7-dihydroxy-2-(4-hydroxyphenoxy)-6-methoxy-4H-chromen-4-one) (41 mg) and 6,7-dimethylesculetin (6,7-dimethoxy-2H-chromen-2-one) (530 mg).
Identification and assessment of the purity of capillin
The identification of capillin and assessment of its purity were accomplished by analysis of NMR spectral data. The [sup.1]H NMR (600 MHz, CD[Cl.sub.3]) and [sup.13]C NMR (150 MHz, CD[Cl.sub.3]) spectra in Fig. 2 were obtained on an FT NMR system (Bruker). [sup.1]H NMR [delta]:8.12 (2H, m, H-2, 6), 7.49 (2H, m, H-3, 5), 7.62 (1H, m, H-4), 2.09 (3H, S, H-6'). [sup.13]C NMR[delta]: 177.2 (C-1'), 136.6, 134.4, 129.6, (C-1-C-6), 86.5, 78.5, 70.8, 63.4 (C-2'-C-5'), 4.93 (C-6').
Analysis of cell death
Apoptotic cells were assessed by an examination of nuclear morphology after staining with Hoechst 33342. Cells were stained with 10 [micro]M Hoechst 33342 for 15 min at 4[degrees]C and were examined under a fluorescence microscope. Analysis of DNA fragmentation by agarose gel electrophoresis was performed as follows: drug-treated or untreated cells were collected by centrifugation and were washed with Dulbecco's phosphate-buffered saline (without [Ca.sup.2+] and [Mg.sup.2+]). The washed cells were suspended in lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS) containing 0.1% RNase A and were incubated for 60 min at 50[degrees]C. The lysate was incubated for an additional 60 min at 50[degrees]C in the presence of 1 mg/ml proteinase K, and the resulting preparation of DNA was analyzed by gel electrophoresis on a 1% agarose gel in Tris acetate buffer (40 mM Tris acetate, 1 mM EDTA). After electrophoresis, DNA was visualized by staining with ethidium bromide.
A suspension of cells was treated with sample compounds. After culturing for 3 days at 37[degrees]C, a solution of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5- carboxanilide (XTT) and phenazine methosulfate (PMS) was added, and the culture was incubated for an additional 4 h. The absorbance at 540 nm was measured on a 96-well plate with a microplate reader.
Preparation of cell lysate
Cells were washed twice with phosphate-buffered saline and lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 5 [micro]g/ml aprotinin, 5 [micro]g/ml leupeptin, 5 [micro]g/ml pepstatin A, 5 [micro]g/ml antipain, 50 mM NaF, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.15 M NaCl, 1% Triton X-100, and 0.5 mM phenylmethylsulfonyl fluoride). The lysate was centrifuged at 15,000 x g for 15 min, and the supernatant was subjected to Western blotting analysis.
Western blotting analysis and assays of protein kinase activities
The cell lysate containing 30 [micro]g of protein was fractionated by SDS-PAGE, and then proteins were transferred to a nitrocellulose membrane. The membrane was first rinsed with TBST (20 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.05% Tween 20) and then blocked with 5% (w/v) skim milk in TBST for 1 h at room temperature. The blocked membrane was subsequently probed for 1 h at room temperature with a 1:200-l: 1000 dilution of first antibodies in blocking buffer. After the membrane had been washed three times with TBST, it was incubated for 1 h at room temperature with either horseradish peroxidase-conjugated antibodies against rabbit IgG or with horseradish peroxidase-conjugated antibodies against mouse IgG. After the membrane had been washed with TBST, bands of protein on the membrane were visualized with an ECL Western blotting detection kit (Perkin Elmer Life Sciences, Inc., Boston, MA). The activities and expression of protein kinases were determined by Western blotting analysis with specific antibodies against p-ERK, ERK, p-JNK, and JNK, respectively.
Analysis of the release of cytochrome c
Cells (1 x [10.sup.6]) were washed with ice-cold Dulbecco's phosphate-buffered saline and were resuspended in 100 [micro]L of ice-cold digitonin lysis buffer (0.02% digitonin in phosphate-buffered saline). After 5 min on ice, the cells were centrifuged at 10,000 x g for 5 min, and the supernatant was subjected to Western blotting analysis with cytochrome c-specific antibody (Vantieghem et al. 1998).
Purification and isolation of capillin
We found strong antitumor activity in the essential oil and aqueous extract of Artemisia capillaris Thunb. flowers with human leukemia HL-60 cells. To clarify the effect of the components of Artemisia capillaris Thunb. flowers on the induction of apoptosis, the major components of the essential oil and aqueous extract were isolated. The essential oil was applied to an HPLC yielding capillin (Fig. 1A) and capillene (Fig. IB), respectively, as described in the 'Materials and methods' section. The aqueous extract was applied to an Amberlite XAD-II column to give [H.sub.2]O and MeOH eluate fractions. The MeOH eluate fraction was subjected to Sephadex LH-20 and repeated silica gel column chromatography to give capillarisin (Fig. 1C) and 6.7-dimethylesculetin (Fig. ID), respectively. We identified capillin by comparing the NMR spectra of the purified compound (Fig. 2A and B) with previously reported spectra (Banerji et al. 1990).
Effects of essential oil on antitumor activity
Essential oils have been reported to have a wide variety of pharmacological activities including antitumor activities (Buhagiar et al. 1999; Paik et al. 2005). Fig. 3 shows the effect of the essential oil from Artemisia capillaris Thunb. flowers on cell viability with human leukemia HL-60 cells. As is evident from the figure, cell viability was decreased by treatment with the essential oil fraction. The concentration of essential oil that decreased cell viability to 50% ([IC.sub.50]) was 3.9 [+ or -] 0.6 [micro]g/ml (Table 1). To evaluate the compounds involved in the induction of antitumor activity, the effect of two compounds isolated from the essential oil fraction on cell viability was investigated. Capillin exhibited potent antitumor activity with an [IC.sub.50] of 6.5 [+ or -] 2.9 [micro]M (Table 1). This growth-inhibitory activity of capillin was much stronger than that of another compound of the essential oil fraction, capillene (Fig. 3). The effect of compounds obtained from the aqueous fraction of Artemisia capillaris Thunb. flowers on cell viability was also examined. For these compounds, cell viability was more effectively decreased by treatment with capillarisin than with 6.7-dimethylesculetin. The [IC.sub.50] values of capillene, capillarisin, and 6.7-dimethylesculetin were 134.9 [+ or -] 16.4 [micro]M, 49.3 [+ or -] 12.2 [micro]M, and 197.4 [+ or -] 17.5 [micro]M, respectively, which is approximately 21-fold, 7.6-fold, and 30-fold less active than capillin, respectively. This difference in antitumor activity between capillin and capillene was presumably due to the presence of the carbonyl group of capillin.
Induction of apoptosis by capillin
Apoptosis was originally defined in terms of characteristic morphological changes (Wyllie et al. 1984), namely, reduction in cell volume, condensation of chromatin in the nucleus, and digestion of chromatin into fragments of DNA in multiples of about 180 bp. To determine whether the features of cell death induced by capillin were from apoptosis or necrosis, the induction of morphological changes by capillin was investigated. Fig. 4 shows morphological changes in HL-60 cells that had been treated with 1 [micro]M capillin for 6 h. The cells developed obvious apoptotic features that included chromatin condensation and nuclear fragmentation (Fig. 4C and D). By contrast, no morphological changes were evident in untreated control cells (Fig. 4A and B). Fig. 4E and F shows the effect of capillene on the induction of apoptotic morphological changes in HL-60 cells. As is evident from the figure, almost no morphological changes were observed under the same experimental conditions (Fig. 4E and F). DNA fragmentation was also analyzed, which is considered to be an early event of apoptosis. Following agarose gel electrophoresis of HL-60 cells that had been treated with 0-5 [micro]M capillin for 8 h, a typical ladder pattern of internudeosomal fragmentation was observed in a dose-dependent manner (Fig. 5A, left panel). However, the extent of DNA fragmentation was inhibited at a higher concentration of 5 [micro]M, which suggests that there might be some intracellular signals that prevent apoptotic events at the higher concentration of capillin. When the cells were treated with 2 [micro]M capillin. the appearance of fragmented DNA was evident within 4 h of beginning the treatment (Fig. 5A, right panel). In contrast, capillene had almost no effect on the induction of DNA fragmentation under the same experimental conditions (Fig. 5B). Fig. 5C shows the effect of capillin on the induction of DNA fragmentation in WI-38 normal human fibroblasts. In contrast to HL-60 cells, treatment of WI-38 cells with capillin did not induce DNA fragmentation. These results indicate that capillin potently initiated apoptosis in human leukemia HL-60 cells.
Effect of capillin on activation of the mitogen-activated protein (MAP) kinase family
Since the MAP kinase family is known to be important in the regulation of cell growth, differentiation, and apoptosis (Davis 2000; Masuda et al. 2000), we examined the effect of capillin on the activation of the MAP kinase family, including ERK1/2 and c-Jun N-terminal kinases (JNK). When HL-60 cells were treated with 1 [micro]M capillin, JNK activity potently increased and reached a maximum after exposure to capillin for 1 h, and no significant alterations in MAP kinase activity were observed (Fig. 6A, left panel). In order to compare the effects of capillin with VP16, an inhibitor of topoisomerase II and a well-known inducer of apoptosis, on activation of the MAP kinase family, HL-60 cells were also treated with VP16 (Walker et al. 1991). VP16 induced a rapid and transient increase in the activity of JNK, while the activity of ERK decreased after treatment with VP16 for 4 h (Fig 6A, right panel). These results indicate that only JNK was significantly activated by capillin, with practically no change in the activation of ERK1/2. Mitochondria play an important role in apoptosis induction via the release of apoptogenic molecules that include cytochrome c (Martinou and Green 2001; Tsujimoto and Shimizu 2000). As demonstrated above, capillin induced apoptotic cell death accompanied by activation of JNK in human leukemia HL-60 cells. Therefore, we next examined the effects of capillin and VP16 on the release of cytochrome c from mitochondria in HL-60 cells. When HL-60 cells were exposed to capillin, a significant increase in the release of cytochrome c within 2-4 h after treatment with capillin was observed (Fig. 6B, left panel). As a positive control, VP16 also enhanced the release of cytochrome c from mitochondria in the cells (Fig. 6B, right panel). These results suggest a correlation between the release of cytochrome c and the induction of apoptosis by capillin.
Artemisia capillaris Thunb. flower has been used in Korea for a wide variety of pharmacological activities. In the present study, we found that the essential oil fraction from Artemisia capillaris Thunb. flower was a potent inhibitor of the growth of cancer cells. A recent report described the biological activity of the essential oils from Artemisia species. The essential oil isolated from Artemisia capillaris Thunb. flower inhibited the expression and production of inflammatory mediators by blocking MAPK-mediated pathways and inhibiting the activation of NF-kappaB and AP-1 (Cha et al. 2009b). The essential oil of Artemisia capillaris Thunb. flower induced apoptosis in human oral cancer cells via mitochondrial stress and caspase activation mediated by the MAPK-stimulated signaling pathway (Cha et al. 2009a). These studies suggested that the essential oil of Artemisia capillaris Thunb. flower contains active components responsible for its pharmacological effect, although the mechanism for this biological action was unclear.
Our findings indicate that among the compounds in the essential oil of Artemisia capillaris Thunb. flower, capillin was the most potent inducer of cell death, as determined using human leukemia HL-60 cells. The death of HL-60 cells induced by capillin was shown to be due to apoptosis as evident by the induction of DNA fragmentation and morphological changes, such as nuclear fragmentation. A recent report described the antitumor effect of capillin on human tumor cells in which capillin was able to induce apoptosis in four human tumor cell lines, colon carcinoma H729, pancreatic carcinoma MIA PaCa-2, epidermoid carcinoma of the larynx HEp-2, and lung carcinoma A549 (Whelan and Ryan 2004). These results are consistent with the results of our present study with human leukemia cells. One feature of the effect of capillin on HL-60 cells was that the concentrations needed to inhibit cell growth and to induce apoptosis were much lower than for other constituents, such as capillene, capillarisin, and 6,7-dimethylesculetin. We have identified several apoptosis inducers in human leukemia HL-60 cells including bufalin (Masuda et al. 1995), geranylgeraniol (Ohizumi et al. 1995), [beta]-hydroxyisovaIerylshikonin (Hashimoto et al. 1999) and sophoranone (Kajimoto et al. 2002). Among these apoptosis inducers, bufalin and [beta]-hydroxyisovalerylshikonin were able to induce apoptosis in HL-60 cells at concentrations lower than [10.sup.-6] M. Upon treatment of HL-60 cells with capillin at 1 [micro]M, apoptosis was strongly induced. This apoptosis-inducing activity was almost the same as that of bufalin and [beta]-hydroxyisovalerylshikonin, suggesting that capillin would also be expected to have potential as an anticancer drug. The [IC.sub.50] value of capillin for HL-60 cells, 6.5 [+ or -] 2.9 [micro]M, was 7.6-30-fold lower than those of the other compounds investigated. Interestingly, the structure of capillin is similar to that of capillene, which lacks a carbonyl group. Since cell growth-inhibition and apoptosis-inducing activities were higher for capillin than for capillene, the differences between the antitumor activity of capillin and capillene might be due to the capillin carbonyl group, which likely plays a role in the induction of apoptosis.
MAP kinase cascades play crucial roles in the induction of apoptosis. Two members of the super family of MAP kinases, JNK/SAPK and p38, which are known as stress-activated protein kinase, are activated by a variety of extracellular apoptotic stimuli (Davis 2000; Masuda et al. 2000). By contrast, activation of ERK, which occurs primarily in response to stimulation by growth factors, prevents apoptosis (Xia et al. 1995). It seems, therefore, that various combinations of inhibition and activation of MAP kinases might be important for induction of apoptosis. Based on the previous report that the essential oil of Artemisia capillaris Thunb. flower induces apoptosis in KB cells through a mitochondria and caspase-dependent mechanism via the MAPK-mediated pathway (Cha et al. 2009a), we investigated the activation of MAPK family proteins in capillin-stimulated HL-60 cells. We found that treatment of human leukemia HL-60 cells with capillin caused preferential activation of one of the stress-activated kinases, JNK, without activation of the growth factor-activated protein kinase, ERK. Although the role of JNK and another type of stress-activated kinase, p38, in the induction of apoptosis by various stimuli has been studied extensively, activation of p38 was not observed after treatment with capillin (data not shown). We also observed a difference between capillin and VP16 in terms of the relationship between the JNK and ERK signaling pathways. VP16 caused downregulation of MAP kinase activity, in addition to activation of JNK activity. Although antitumor activity of VP16 was higher than that of capillin in human leukemia cells (Table 1), the signal transduction cascade induced by capillin was different from VP16 as described above, suggesting that capillin might have a selective effect on induction of apoptosis in various other cancer cells. A recent report described that the antitumor activity of capillin was much more potent in epidermoid carcinoma of the larynx HEp-2 cells than in other cancer cells, such as colon carcinoma HT29, pancreatic carcinoma MIA PaCa-2, and lung carcinoma A549 (Whelan and Ryan 2004). These results suggest that capillin might be a unique candidate for an anticancer drug.
In addition to the MAP kinase cascade, the release of cytochrome c plays an important role in the induction of apoptosis (Martinou and Green 2001; Tsujimoto and Shimizu 2000). Cytochrome c release from mitochondria is a key mechanism in the initiation of apoptosis. Upon treatment of cells with various inducers of apoptosis, cytochrome c is released from intermembrane spaces of mitochondria into the cytoplasm, with resultant activation of caspase-9 by association with Apaf-1 and deoxy ATP or ATP (Li et al. 1997).
Subsequently, activated caspase-9 triggers apoptotic cell death by activating an effector caspase, caspase-3. Previously, we purified the compound that induced apoptosis in human leukemia cells and identified it as the flavonoid sophoranone (Kajimoto et al. 2002). Cytochrome c was released upon treatment of isolated mitochondria with sophoranone, suggesting that sophoranone is a unique apoptosis-inducing anticancer agent that targets mitochondria. In the present study, capillin induced apoptosis with the resulting release of cytochrome c from mitochondria in HL-60 cells. The molecular mechanism responsible for cytochrome c release in response to capillin remains to be elucidated. We are currently investigating this mechanism of cytochrome c release and the relationship between apoptosis induction and the activation of JNK.
In conclusion, our results introduce the possibility that capillin might be a useful anticancer drug that could enhance therapeutic effectiveness in conjunction with other conventional anticancer drugs. Further work is needed to reveal the details of the molecular mechanism of capillin-induced apoptosis.
Conflict of interest
This work was supported in part by Grants-in-Aid (no. 19-8) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The authors would also like to thank all of the researchers who were involved in the research.
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Yutaka Masuda (a), *, Keisuke Asada (a), Rei Satoh (b), Kimihiko Takada (a), Junichi Kitajima (b)
(a) Laboratory of Clinical Pharmacy, Showa Pharmaceutical University, Higashi Tamagawa Gakuen, Machida, Tokyo 194-8543, Japan
(b) Laboratory of Kampo Medicinal Education, Showa Pharmaceutical University, Higashi Tamagawa Gakuen, Machida, Tokyo 194-8543, Japan
Received 17 July 2014
Revised 11 March 2015
Accepted 12 March 2015
Abbreviations: Caspase, cysteine-dependent aspartate-directed protease; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; VP16, etoposide.
* Corresponding author. Tel.: +81 42 721 1560; fax: +81 42 721 1560.
E-mail address: firstname.lastname@example.org (Y. Masuda).
Table 1 Antitumor activities of compounds isolated from Artemisia capillaris Thunb. flower. Compounds [IC.sub.50] ([micro]g/ml) VP16 0.5 [+ or -] 0.3 6,7-Dimethylesculetin 40.7 [+ or -] 3.6 Capillarisin 15.7 [+ or -] 3.9 Essential oil 3.9 [+ or -] 0.6 Capillene 18.9 [+ or -] 2.3 Capillin 1.1 [+ or -] 0.5 Compounds [IC.sub.50] ([micro]M) VP16 0.85 [+ or -] 0.5 6,7-Dimethylesculetin 197.4 [+ or -] 17.5 Capillarisin 49.3 [+ or -] 12.2 Essential oil ND Capillene 134.9 [+ or -] 16.4 Capillin 6.5 [+ or -] 2.9 HL-60 cells were treated with each compound for 3 days. IC50 is the concentration that gave 50% inhibition of growth. Percentages of viable cells were determined by the XTT assay as described in the 'Materials and methods' section. All results shown are means [+ or -] S.D. from four independent experiments. ND: not determined.
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|Author:||Masuda, Yutaka; Asada, Keisuke; Satoh, Rei; Takada, Kimihiko; Kitajima, Junichi|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||May 15, 2015|
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