Scopadulciol, Isolated from Scoparia dulcis, Induces β-Catenin

Mar 20, 2015 - Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 ... Pharmacy Discipline, Life Science School, Khulna University, Kh...
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Scopadulciol, Isolated from Scoparia dulcis, Induces β‑Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells Rolly G. Fuentes,†,⊥ Kazufumi Toume,† Midori A. Arai,† Samir K. Sadhu,‡ Firoj Ahmed,§ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh § Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh ⊥ University of the Philippines Visayas Tacloban College, 6500 Tacloban City, Philippines ‡

S Supporting Information *

ABSTRACT: Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2−4) by an activity-guided approach using the TCF reporter (TOP) luciferasebased assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL. β-catenin/TCF signaling activity was shown to be upregulated in most gastrointestinal cancer cells.8 These tumor cells accumulate β-catenin due to mutations in the destruction complex, such as APC, or in β-catenin itself. The use of tumor necrosis factor-related apoptosis ligand (TRAIL) to induce cancer cell apoptosis has been considered an attractive strategy because it targets cancer cells but not normal cells.9 When TRAIL binds to death receptors (DR4 and DR5), it activates an effector caspase (caspase-3) by an initiator caspase (caspase-8).10 TRAIL-induced apoptosis also requires the mitochondrial pathway to completely activate caspase-3.11 However, several cancer cells have developed resistance to the effects of TRAIL due to the overexpression of decoy receptors and antiapoptotic proteins such as Bcl-2 and IAPs (e.g., survivin).12 Treatments that combine TRAIL with compounds that can either upregulate the expression of DR4 or DR5 or downregulate antiapoptotic protein levels represent

β-Catenin is an evolutionarily conserved protein that is a major structural component of the cadherin-based adherens junction and also plays a key role in Wnt signaling.1 β-Catenin-regulated Wnt signals have been shown to regulate cell development and proliferation, as well as tissue homeostasis.2 In normal cells and in the absence of the Wnt protein, β-catenin is maintained at low levels through the actions of a destruction complex composed of axin, adenomatous polyposis coli (APC), glycogen synthase kinase3β (GSK3β), and casein kinase1α (CK1α). The kinases (GSK3β and CK1α) phosphorylate β-catenin,3 and the β-transducin repeat-containing protein (β-TrCP) then recognizes phosphorylated β-catenin for ubiquitination and proteasomal degradation. 4 The destruction complex is inactivated in the presence of the Wnt protein due to the saturation of phosphorylated β-catenin.5 Cytosolic β-catenin accumulates, translocates to the nucleus, and activates T-cell factor/lymphoid enhancing factor (TCF/LEF) for the transcription of target genes such as c-myc and cyclin D1.6,7 However, a dysregulation in β-catenin levels may lead to the development of various human diseases such as cancer.2 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 8, 2015

A

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screening of our plant extract library using the TOP assay. Based on the screening results, the MeOH extract of S. dulcis (aerial part) inhibited TOP activity by 82% at 10 μg/mL. The viability of STF/293 reporter cells was 80% at the same concentration. An activity-guided approach was used to identify active compounds. The purification of the compounds by silica gel, ODS column chromatography, and HPLC afforded two diterpenes, scopadulciol (1)16 and scopadiol (2),18 and two triterpenes, friedelin (3) and glutinol (4). The TOP assay utilized STF/293 cells stably transfected with the SuperTOPflash plasmid, which contained wild-type TCF binding sites. GSK3β activity was inhibited upon addition of LiCl, which increased the stability of β-catenin.19 Accumulated β-catenin then translocated to the nucleus and formed a complex with TCF to initiate transcription of the luciferase gene. To eliminate nonspecific TOP inhibition, the FOP assay was conducted by transiently transfecting HEK293 with the SuperFOPflash plasmid, which contained mutations in its TCFbinding site and was nonresponsive to active Wnt signals. Of the four compounds isolated, only 1 inhibited TOP activity. However, 1 also decreased FOP activity, indicating that the 1-induced inhibition of TOP activity was not specific to TOP (Figure 1).

potential strategies to overcome TRAIL resistance in cancer cells. The downregulation of β-catenin has been suggested as another possible strategy to enhance TRAIL-mediated apoptosis in resistant cancer cells.13 In our continuing efforts to search for natural compounds with potent inhibitory activities, we conducted a screening program of plant extracts collected from Bangladesh using a cell-based luciferase assay to evaluate TCF/β-catenin transcription activity. Our screening assay utilized HEK293 cells that were stably transfected with the TCF reporter gene. Several natural compounds that inhibit the Wnt signal have been identified by our laboratory using this screening system, such as the limonoid xylogranin B, which was isolated from Xylocarpus granatum.14 Calotropin, a cardenolide isolated from Calotropis gigentea, was previouly shown to exhibit potent Wnt inhibitory activity by enhancing CK1α.15 In this study, Scoparia dulcis L. (Plantaginaceae) was identified as a potential source of Wnt inhibitors because it stronly inhibited TOP activity. Although activity-guided isolation using this assay led to the isolation of four compounds (1−4), only scopadulciol (1) exhibited inhibitory activity against the Wnt signal. Previous studies demonstrated that 1 exhibited inhibitory activity against gastric H+,K+-ATPase, which is responsible for acid secretion in the stomach,16 and cytotoxicity against several gastric cancer cell lines.17

Figure 1. Columns show the TOP and FOP activities of 1. TOP was measured using HEK293 cells stably transfected with the TOP reporter gene (STF/293), while FOP activity was measured using HEK293 cells transiently transfected with the FOP reporter gene. The line graph represents the viability of HEK293 cells. Quercetin was used as the positive control in the TOP assay. Data are from one experiment representative of at least two independent experiments (mean ± sd).

Compound 1 was previously shown to exhibit cytotoxic activity against several gastric cancer cell lines;17 however, its mechanism of action had yet to be elucidated. In the present study, 1 decreased the viability of the gastrointestinal cancer cells SW480 (colon), HCT116 (colon), DLD1 (colon), and AGS (gastric) cells (human), which were previously reported to have an active Wnt signal, and RKO (colon) cells, the proliferation of which is known to be independent of β-catenin (Figure 2 and Figure S1a, Supporting Information). Compound 1 was also partially cytotoxic to the non-cancer-derived HEK293 cells. The viability assay using FMCA showed that 1 greatly decreased the viability of RKO and AGS cells. Compound 1 was more cytotoxic to RKO cells (IC50 = 359 nM) than to AGS cells (IC50 >500 nM) after 24 h (Table 1). However, RKO viability increased after 48 h, whereas AGS viability decreased (Figure 2a). In addition, a cell cycle analysis revealed that 1 increased the G0/G1 population in RKO cells after 24 h (Figure 2b). On the other hand, the treatment of AGS cells with 1 resulted in a dose-dependent increase in the sub-G1 cell population, suggesting that 1 may have induced apoptosis. No significant change was observed in the sub-G1

We herein demonstrate that 1 exhibits antiproliferative activity against gastrointestinal cancer cells, and this may be due to the proteasome-dependent degradation of β-catenin and decreases in the levels of cyclin D1, c-myc, and survivin. Also, the potential of 1 to overcome TRAIL resistance in AGS human gastric adenocarcinoma cells was also investigated. The results obtained revealed that the combined treatment with TRAIL and 1 had an additive effect and increased apoptosis in AGS human gastric adenocarcinoma cells. To the best of our knowledge, this is the first study on the mechanism of action of 1 in the cell death of gastric cancer cells.

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RESULTS AND DISCUSSION Scopadulciol (1) Decreased the Viability of AGS. S. dulcis was identified as a potential source of Wnt inhibitors during the B

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Figure 2. (a) Effects of 1 on the viabilities of RKO and AGS cells. Cells were treated with 1 at different concentrations (125, 250, and 500 nM) for 24 h. Viability was determined using the FMCA and was normalized to day 0. (b) Effects of 1 on cell cycles in RKO (upper panel) and AGS cells (lower panel). Cells were exposed to 1 (150 and 300 nM) for 24 h and then stained with propidum iodide, as described in the Experimental Section. The distribution of cells in different phases was obtained using a FACS analysis. Data are from one experiment representative of at least two independent experiments.

by Western blot analysis using AGS cells. As shown in Figure 3a, 1 decreased β-catenin levels in AGS cells in a concentrationdependent manner after a 24 h exposure. Furthermore, it inhibited the accumulation of β-catenin in the nucleus, as shown by decreases in nuclear β-catenin levels. In the Wnt signaling pathway, the phosphorylation of β-catenin by CK1α and GSK3β was previously identified as an important step in the ubiquitin/proteasome-mediated degradation of β-catenin.3,4 However, we observed that the level of GSK3β (Figure S2a, Supporting Information) as well as that of phosphorylated β-catenin (Figure S2b, Supporting Information) was decreased by 1. If the degradation of β-catenin is modulated in a GSK3βdependent manner, the decreases in GSK3β and phosphorylated β-catenin levels may cause an increase in β-catenin levels. These results suggested that the degradation of β-catenin by 1 was independent of GSK3β. β-Catenin Degradation Induced by Scopadulciol (1) Was Proteasome-Dependent. The mutation of β-catenin (G34E) in AGS cells was previously suggested to reduce the efficiency of the ubiquitin-proteasome degradation of β-catenin by affecting the GSK3β phosphorylation motif, which is important for the ubiquitination process.8 To determine whether 1 could induce the proteasome-mediated degradation of β-catenin in AGS, AGS cells were pretreated with either MG132 or epoxomicin for 1 h and then incubated with 1 for 24 h.

Table 1. Cytotoxic Activity of 1 against Human Cell Lines IC50a [nM] time (h)

SW480

24 48 72

>500 >500 296

b

HCT116 >500 >500 >500

b

DLD1b

AGSb

RKOb

HEK293c

>500 >500 >500

>500 101 70

359 374 479

>500 227 181

a

Inhibitory concentration that causes 50% cell death. bGastroinstestinal cancer cell lines. cNon-cancer-derived cell line.

cell population in RKO even at 300 nM. Since 1 induced apoptosis in AGS, these cells were utilized in the subsequent experiments in order to investigate the possible mechanism responsible for the cytotoxic activity of 1. The cytotoxic activity of 1 against AGS cells was not abolished in the presence of z-VAD-fmk, a pan caspase inhibitor (Figure S1b, Supporting Information), indicating that the cytotoxic activity of 1 did not involve caspase activity. The putative mode of action by which 1 could induced cytotoxicity against the AGS cancer cell line was further investigated. Scopadulciol (1) Decreased β-Catenin Levels in AGS. AGS cells have been shown to have an active Wnt signaling pathway due to mutations in β-catenin.8 Although the TOP/FOP assay suggested that inhibition was not specific to TOP in STF/293 cells, we further verified the effects of 1 on Wnt signaling C

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Figure 3. Western blot analysis on (a) level and localization of β-catenin in AGS cells treated with different concentrations of 1 (100, 200, and 400 nM) for 24 h. Whole cell, cytoplasmic, and nuclear lysates were obtained from AGS cells and subjected to Western blot analysis with an anti-βcatenin antibody. β-actin served as protein control for the full and cytoplasmic fractions while histone H1 served as the control for the nuclear fraction. Effects of the MG132 (b) and epoxomicin (c) pretreatments on β-catenin levels in AGS cells treated with 1 (400 nM). Cells were pretreated with either MG132 (10 μM) or epoxomixin (10 μM) for 1 h and then treated with 1 (400 nM) for 24 h. Effects of 1 on the level of p53 (d) and β-catenin in AGS treated for 24 h in the absence or presence of pifithrin-α (20 μM) (e). The whole cell lysate was obtained and analyzed by Western blotting. (f) Effects of pifithrin-α (20 μM) on the viability of AGS treated with 1 for 48 h. The viability of AGS was measured using FMCA.

of GSK3β and β-TrCP, via the Siah-1 mechanism.24 Western blot analysis showed that 1 increased the level of p53 (Figure 3d). The role of p53 in the 1-induced degradation of β-catenin was then investigated by treating AGS cells with 1 in the absence or presence of pifithrin-α, an inhibitor of p53 transcriptional activity.25 Western blot analysis showed that pifithrin-α abolished the degradation of β-catenin by 1, while β-catenin levels in AGS cells treated with pifithrin-α alone remained unaffected (Figure 3e). These results suggested that p53 mediated the degradation of β-catenin induced by 1. As discussed above, mutation in β-catenin in AGS has been shown to affect the GSK3β phosphorylation motif, which is important for the ubiquitination process.8 Since proteasome degradation via β-TrCP is inefficient in AGS, 1 may have affected another mechanism that can induce the degradation of β-catenin. Other than the phosphorylation-dependent degradation of β-catenin, the Siah-1 mechanism promoted degradation of β-catenin independent of GSK3β and β-TrCP.24,26 In this mechanism, Siah-1, which can be induced by p53, interacts with

MG132 and epoxomicin are proteasome inhibitors.20 The treatment with MG132 or epoximicin alone did not significantly affect β-catenin levels. The pretreatment with MG132 or epoximicin abolished the degradation of β-catenin, in AGS cells treated with 1, indicating that the 1-induced degradation of β-catenin was proteasome-dependent (Figure 3b,c). β-Catenin has also been reported to be a substrate of the proteases caspase21,22 and calpain;23 therefore, the participation of these proteases in β-catenin degradation was also determined. AGS cells were pretreated with the pan-caspase inhibitor z-VAD-fmk or with the calpain inhibitor calpastatin for 1 h and then treated with 1. The pretreatment with the protease inhibitors did not protect against the degradation of β-catenin by 1 (Figure S2c,d, Supporting Information). This result indicated that neither caspase nor calpain participated in the degradation of β-catenin induced by 1. Degradation of β-Catenin by Scopadulciol (1) and Its Cytotoxic Activity in AGS Involved p53 Activity. p53 was previously shown to decrease the stability of β-catenin, independent D

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determined by Western blot analysis. Figure 4b shows that 1 decreased the levels of cyclin D1, c-myc, and survivin in a dosedependent manner, further suggesting that 1 inhibited TCF/ β-catenin transcriptional activity in AGS cells. These results suggested that the degradation of β-catenin induced by 1 resulted in the inhibition of TCF/β-catenin transcriptional activity in AGS cells. Decreases in the nuclear accumulation of β-catenin by 1 may also have resulted in the inhibition of TCF/β-catenin transcriptional activity in AGS. Since cyclin D1, c-myc, and survivin are involved in cell cycle progression and proliferation,28−30 the inhibition of the Wnt signals may be associated with the cytotoxic activity of 1 in AGS. As discussed above, the 1-induced degradation of β-catenin in AGS was mediated by p53. The inhibition of p53 activity resulted in a reduction in the cytotoxic effects of 1 in AGS. This result supports the participation of inhibited Wnt signal in the cytotoxic activity exhibited by 1 in AGS cells. Scopadulciol (1) Sensitized AGS to TRAIL-Induced Apoptosis. The potential of scopadulciol to overcome TRAIL resistance in AGS was also investigated. In this study, AGS cells resisted apoptosis at TRAIL concentrations up to 100 ng/mL, which only decreased viability to 86% (Figure 5a). When TRAIL was administered with luteolin31 (17.7 μM), which was used as a positive control, viability was further decreased to 41%. The treatment of AGS with 1 alone resulted in a slight decrease in cell viability (70% at 200 nM). However, when 1 was combined with TRAIL, they synergistically exhibited stronger cytotoxic activity on AGS than the treatments with TRAIL or 1 alone (Figure 5a). The combined treatment with 1 (200 nM) and TRAIL (100 ng/mL) markedly reduced AGS viability to 7% even after only a 24 h exposure. The induction of apoptosis was also investigated by the annexin V-affinity assay. The staining of cells with annexin-V and propidium iodide can discriminate between live, apoptotic, and dead cells.32 The FACS analysis revealed that the treatment of AGS cells with combined 1 (200 nM) and TRAIL (100 ng/mL) induced apoptosis more (27.7% apoptotic cells) than the treatment with TRAIL (5.7%) or 1 alone (3.5%) as early as 12 h (Figure 5b). On the other hand, no significant differences were observed in the number of apoptotic cells in AGS between either TRAIL or 1 alone and the control. These results suggested that 1 exhibited strong activity to overcome TRAILresistance. Furthermore, the treatment of the non-cancer-cell line HEK293 with combined 1 and TRAIL led to lower reductions in viability (Figure 5c) than AGS. This result indicated that the combined treatment with 1 and TRAIL was preferentially toxic to AGS cancer cells over HEK293 non-cancer cells. Scopadulciol (1) Affected the Levels of ApoptosisRelated Proteins. The effects of 1 on Bcl-2 and the death receptors, DR4 and DR5, were determined in order to identify the mechanism involved in the activity of 1 to overcome TRAIL-resistance (Figure 6a). Western blot analysis revealed that 1 increased DR4 and DR5 levels and decreased the level of the antiapoptotic protein Bcl-2. The increases in the DR4 and DR5 levels may have enhanced the binding of TRAIL to the death receptors. The binding of TRAIL to death receptors has been shown to activate caspase-8, which then activates caspase3 to mediate apoptosis.10 Moreover, the mitochondrial pathway further enhanced 1 + TRAIL-induced apoptosis as 1 decreased Bcl-2 and survivin levels. Bcl-2 is known to inhibit the proapoptotic protein Bax, while survivin inhibits the activation of procaspase-9 to caspase-3.11 The increased binding of TRAIL to the death receptors and decreased levels of the antiapoptotic

APC and promotes the ubiquitination and proteasomal degradation of β-catenin.24,26 Since the 1-induced degradation of β-catenin was shown to be mediated by p53, the Siah-1 mechanism may be involved when β-catenin is ubiquitinated for its proteasomal degradation. The action of 1 on the degradation of β-catenin in AGS may also be cell-dependent because decrease in β-catenin levels was not observed in SW480 or STF/293 cells (data not shown). Since p53 mediated the degradation of β-catenin by 1, the participation of p53 activity in the cytotoxic activity of 1 was then studied. A previous study reported that p53 could induce either cell survival or apoptosis depending on the type of tissue and cellular environment.27 AGS cells were treated with 1 in the absence or presence of pifithrin-α. Pifithrin-α abrogated the cytotoxic activity of 1 in AGS after 48 h (Figure 3f). This result suggested that 1 exhibited p53-dependent cytotoxic activity on AGS cells. Scopadulciol (1) Downregulated the Expression of Wnt/β-Catenin Target Proteins in AGS. As discussed above in Figure 1, the results of the TOP/FOP assay using HEK293 cells suggested that 1 may not have inhibited TCF/β-catenin transcriptional activity. However, the level of β-catenin was decreased in AGS cells and its accumulation in the nucleus was also inhibited by 1, implying that this may have inhibited TCF/β-catenin transcriptional activity. AGS cells were transfected with either SuperTOPflash or SuperFOPflash plasmids and then incubated with 1. As shown in Figure 4a, the

Figure 4. (a) TOP and FOP activities of 1 in AGS cells. AGS cells were cotransfected with either the TOP or FOP reporter gene and pRL-CMV plasmids for 24 h. After this transfection, cells were incubated with 1. Luciferase activities were measured at 24 h posttransfection. Data were representative of one experiment from at least two independent experiments (mean ± sd). (b) Western blot analysis using full lysate on the levels of β-catenin/TCF target proteins in AGS cells after treatments with different concentrations (100, 200, or 400 nM) of 1 for 24 h. The whole cell lysate was obtained and subjected to Western blot analysis with anti-c-myc, anti-cyclin D1, and anti-survivin antibodies. β-actin served as a protein control.

treatment with 1 resulted in a decrease in TOP activity without significantly affecting FOP activity. This result indicated that 1 inhibited TCF/β-catenin transcriptional activity in AGS cells. The expression levels of the β-catenin-dependent target proteins cyclin D1,28 c-myc,29 and survivin30 were then E

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Figure 5. (a) Compound 1 showed activity to overcome TRAIL-resistance in AGS cells. Cells were exposed to different treatments as described for 24 h and assayed for viability using the FMCA. Lut = Luteolin served as a control for the assay of activity to overcome TRAIL-resistance. (b) FACS analysis on the induction of apoptosis in AGS cells by the 1 + TRAIL combined treatment. Cells were exposed to the different conditions as described for 12 h. Cells were collected and stained with annexin V and propidium iodide. The populations of viable, apoptotic, and dead cells were quantified by a FACS analysis. (c) Effects of combined treatment of TRAIL and 1 against non-cancer-derived HEK293 cells. Cells were exposed to the different treatments as described for 24 h. Viability was determined using the FMCA and was normalized to that of the control (0 nM).

β-catenin decreased TRAIL sensitivity by lowering the expression of DR4 and DR5 in colon cancer cells.36 Since the function of the Wnt/β-catenin signaling pathway is to mediate cancer cell growth and survival, the degradation of β-catenin by 1 may, at least partly, contribute to the action of TRAIL in inducing apoptosis. Scopadiol (2), which is a structurally related compound and was also isolated during the activity-guided isolation process, exhibited TOP inhibitory activity at a higher concentration (IC50 45 μM) (Figure S3a, Supporting Information). However, it did not have antiproliferative (Figure S3b, Supporting Information) activity or activity to overcome TRAIL-resistance in AGS cells (Figures S3c, Supporting Information). Based on the structures of 1 and 2, the additional two cyclic groups in 1 may be important for its activity. These results may provide information for future studies on structural modifications to 1 in order to make it more active and less cytotoxic to normal cells. In conclusion, this study demonstrated that 1 suppressed Wnt signaling in AGS by inducing the p53- and proteasomemediated degradation of β-catenin and inhibiting the accumulation of β-catenin in the nucleus. The inhibitory activity of 1 against the Wnt signal might have contributed to its cytotoxic activity in AGS. We also showed that 1 could sensitize AGS to TRAIL-induced apoptosis by increasing DR4 and DR5 levels and downregulating Bcl-2. The degradation of β-catenin by 1 in AGS may also have facilitated TRAIL resistance being overcome and warrants further investigations.

proteins may have mediated AGS to overcome its resistance to TRAIL-induced apoptosis. To determine whether the decrease in viability by 1 + TRAIL was triggered by caspases, AGS cells were pretreated with z-VAD-fmk. The results of the viability assay showed that z-VAD-fmk blocked apoptosis induced by the combined treatment of 1 and TRAIL (Figure 6b), suggesting that the effects of combined 1 and TRAIL on the cell viability of AGS may have been caspase-dependent. A further decrease in β-catenin levels in AGS treated with a combination of 1 and TRAIL was also observed in this study (Figure 6c). Decreases in β-catenin levels were also reported when AGS cells were treated with TRAIL and the natural compound sanguinarine.33 Furthermore, their study showed that the activation of caspase by the combined treatment of TRAIL and sanguinarine caused the cleavage of β-catenin. Western blot analysis was then used to determine whether the degradation of β-catenin by 1 + TRAIL was also mediated by caspase. In contrast to previous findings, the pretreatment of AGS with z-VAD-fmk did not prevent 1 + TRAIL-induced β-catenin degradation, which suggested that caspases did not participate in the degradation of β-catenin (Figure 6d). The direct relationship between decreased βcatenin levels and TRAIL-mediated apoptosis remains unclear. A previous study had identified osteoprotogerin (OPG) as a decoy receptor of TRAIL, which can block the TRAIL-induced apoptosis.34 Furthermore, it was also shown that the expression of OPG is regulated by the Wnt/β-catenin signal and mediates TRAIL resistance in colon cancer cells.35 Thus, Wnt signal inhibition may be one mechanism to overcome TRAIL-resistance. However, a previous study also showed that the downregulation of

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EXPERIMENTAL SECTION

General Experimental Procedures. Silica gel PSQ100B and Sephadex LH-20 (GE Healthcare, Little Chalfont, UK) were used for column chromatography, Preparative HPLC was performed using F

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Figure 6. (a) Western blot analysis of DR4, DR5, and Bcl-2 in AGS treated with different concentrations of 1. Cells were treated with 1 at different concentrations (50, 100, and 200 nM) for 24 h and analyzed by Western blotting. (b) Effects of z-VAD-fmk (50 μM) on the viability of AGS cells treated with combined TRAIL and 1. Cells were pretreated with z-VAD-fmk (50 μM) for 1 h, then treated as described for 24 h. Viability was calculated relative to that of the control (no inhibitor and TRAIL) treatment. Data were representative of one experiment from at least two independent experiments (mean ± sd). (c) Western blot analysis of β-catenin in AGS cells treated with combined treatment 1 (200 nM) + TRAIL (100 ng/mL) for 12 h. (d) Effects of the z-VAD-fmk pretreatment on β-catenin levels in AGS treated with combined 1 (200 nM) + TRAIL (100 ng/mL). Cells were pretreated with z-VAD-fmk (50 μM) for 1 h and treated with 1 (400 nM) + TRAIL (100 ng/mL). The whole cell lysate was obtained after a 12 h exposure and was then subjected to Western blot analysis with an anti-β-catenin antibody. β-actin served as protein control. Cosmosil 5C18−AR−II and Cholester (Nacalai Tesque Inc., Kyoto, Japan). 1H and 13C NMR spectra were recorded on JEOL JNM-ECA600 spectrometers with deuterated solvent. HRESIMS were measured on a JEOL JMS-T100LP spectrometer. Optical rotation was determined with a JASCO P-1020 polarimeter. STF/293 cells were a generous gift from Prof. Jeremy Nathans (John Hopkins Medical School); AGS, SW480, HCT116, DLD1 RKO, and HEK293 cells were purchased from ATCC. STF/293, HEK293, SW480, DLD1, and HCT116 were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) (Wako, Osaka, Japan) with 10% FBS, while AGS cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Wako) with 10% FBS. Cultures were maintained in a humidified incubator at 37 °C in 5% CO2/95% air. Plant Material. The aerial parts of Scoparia dulcis (Plantaginaceae) were collected at Khulna in Bangladesh on January, 2011, and identified by the experts of Forestry and Wood Technology Discipline, Khulna University, Bangladesh. A voucher specimen (KKB304) was deposited in the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Extraction and Isolation. The dried aerial parts of S. dulcis (128 g) were soaked in MeOH and left overnight at room temperature, homogenized, filtered, and evaporated under reduced pressure to obtain a crude extract (3.0 g). The chlorophyll content of the MeOH crude extract was removed by Diaion HP-20 column chromatography (φ 40 mm × 230 mm) using acetone/MeOH (0:1−1:0) and yielded three fractions (1A−1C). Fraction 1B (0.5 g), which was eluted with 100% MeOH and showed a potent decrease in TOP activity (10 μg/mL, 91%), was subjected to silica gel column chromatography (φ 40 mm × 275 mm) and eluted with the CHCl3/MeOH gradient system (1:0 to 0:1) to give fractions 2A−2K. Active fraction 2G (281.4 mg), which was eluted with CHCl3/MeOH 95:5, was subjected to silica gel column chromatography (φ 40 mm × 280 mm) using the CHCl3/MeOH gradient system (9:1 to 0:1) to give fractions 3A−3L. Fraction 3J

(25.8 mg), which was eluted with CHCl3/MeOH (7:3), was further purified by preparative HPLC [Cosmosil 5C18-AR-II; φ 10 mm × 250 mm; 85% MeOH; 2 mL/min] to give compound 1 (5.6 mg, tR 20.4 min). Fraction 1A (2.2 g), which was eluted with 100% MeOH, was resuspended in 10% MeOH (300 mL) and partitioned between hexane, EtOAc, and n-BuOH (300 mL × 3). The hexane fraction was then subjected to Sephadex LH-20 column chromatography (φ 25 mm × 250 mm) with MeOH to give fractions 5A−5E. The purification of fraction 5D (15.7 mg) by HPLC [Cosmosil Cholester; φ 10 mm × 250 mm; 80% MeOH; 3 mL/min] yielded fractions 6A−6D. Fraction 6B was identified as compound 2 (1.3 mg, tR 21.2 min). Fraction 6A (1.8 mg) was further purified by silica gel column chromatography (φ 5 mm × 60 mm) and yielded compound 1 (1.5 mg). The purity of 1 was determined to be ≥95% based on HPLC and 1H NMR. The recrystallization of fractions 2D (2.6 mg) and 2E (11.7 mg) in CHCl3/MeOH yielded compounds 3 (0.8 mg) and 4 (0.5 mg), respectively. Reporter Gene Assay and Transfection. TCF/β-catenin transcriptional activity was determined using a previously described method.19 The screening assay (TOP assay) utilized HEK293 cells, which were stably transfected with the SuperTOPflash plasmid (STF/ 293). STF/293 cells were seeded into 96-well plates (3 × 104 cells/ well) and, after a 24-h incubation, treated with compounds combined with 15 mM LiCl. After 24 h, cells were lysed, and luciferase activity was measured. In the control reporter assay (FOP assay), HEK293 cells were briefly split into 24-well plates (1 × 105 cells/well) and incubated for 24 h. These cells were then transfected with 500 ng/well of the luciferase reporter construct (SuperFOPflash) and 50 ng/well of pRL-CMV (Promega, USA) for normalization. The compounds combined with 15 mM LiCl were added at 12 h post-transfection. To determine TCF/β-catenin transcriptional activity in AGS cells, 1 × 105 cells were seeded onto 24-well plates, incubated for 24 h, and then transfected with either SuperTOPflash or SuperFOPflash (500 ng/well) and pRL-CMV (25 ng/well) for 24 h. The transfected cells were then G

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period. These cells were harvested and washed using the same process as described in the cell cycle analysis. They were then stained with PI and the Annexin-V-fluos staining kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol and run on Millipore Guava Easycyte5. Data were analyzed using Guava InCyte software (Millipore).

treated with the compound for 24 h. Cells were lysed after being incubated for 24 h, and luciferase activity was measured using PICAGENE Dual Sea Pansy (Toyo Ink, Tokyo, Japan). Cell Proliferation Assay. Cell viability was measured using a fluorometric microculture cytotoxicity assay (FMCA).37 STF/293 (3 × 104 cells/well), AGS, SW480, HCT116, DLD1, and RKO (5 × 103 cells/well) were inoculated onto 96-well plates for 24 h. Compounds were then added and incubated for different time periods. After being incubated, they were treated with fluorescein diacetate (0.35 mg/mL) (Wako, Japan) in PBS buffer. Fluorescence was detected after a 1 h incubation. In the time-dependent experiments, relative cell viability was normalized to 0 d (24 h after cell seeding). Assay of Activity To Overcome TRAIL-Resistance. The activity to overcome TRAIL-resistance was determined using a previously described method.38 AGS cells were seeded on 96-well plates (6 × 103 cells/well), incubated for 24 h, and then treated with different concentrations of the compounds, either alone or in combination with TRAIL (100 ng/mL). Viability was assessed after 24 h using the FMCA method described above. Western Blot Analysis. Whole cell extracts were prepared as described previously.14 Cytoplasmic and nuclear fractions were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s recommendations. Proteins were separated on 7.5−12.5% SDSpolyacrylamide gels and then transferred onto a nitrocellulose transfer membrane (Pall) or onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk in TBST and probed with anti-βcatenin (1:2000, BD Biosciences, no. 610153, San Jose, CA, USA), anti-c-myc (1:200, Santa Cruz Biotechnology, no. sc-40, Santa Cruz, CA, USA), anti-cyclin D1 (1:1000, Abcam, no. ab6152, Cambridge, MA, USA), anti-survivin (1:500, Cell Signaling Technology, no. 71G4B7, Danvers, MA, USA), anti-p53 (1:500, Sigma-Aldrich, no. P5813, St. Louis, MO, USA St. Louis, MO, USA), anti-Bcl-2 (1:1000, Sigma-Aldrich, no. B9804), anti-TNFRSF10A/DR4 (1:500, SigmaAldrich, no. WH0008797M1), anti-DR5 (1:500, Sigma-Aldrich, no. D3938), anti-histone H1 (1:1000, Santa Cruz Biotechnology, no. sc8030), and anti-β-actin (1:4000, Sigma-Aldrich, no. A2228) primary antibodies overnight at 4 °C. The anti-β-actin antibody served as an internal control for whole and cytoplasmic lysates while anti-histone H1 served as the control for the nuclear lysate. Membranes were then washed with TBST and incubated with either horseradish peroxidase conjugated anti-mouse IgG (1:4000, GE Healthcare, no. NA931VS, Little Chalfont, U.K.) or anti-rabbit (1:4000, Jackson Immuno Research, no. 705-035-003, West Grove, PA, USA) for 1 h at room temperature. After the membranes were washed with TBST, protein bands were visualized using the ECL Advance Western Blotting detection system (GE Healthcare) or Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) and captured with Molecular Imager ChemiDoc XRS+ (Bio-Rad Laboratories). Cell Cycle Analysis by Flow Cytometry. The distribution of cells, with or without the treatment, in the different phases of the cell cycle was estimated by measuring cellular DNA using flow cytometry. A random population of AGS cells was seeded on 12-well plates (1 × 105 cells/well) for 24 h and treated as described. The medium containing floating cells was collected, and attached cells were washed with PBS and then detached with trypsin. All the washings, medium, and detached cells were combined and centrifuged at 1000 rpm for 5 min, and the cells were then washed twice with PBS. These cells were resuspended in 0.3 mL of PBS and fixed in 70% ethanol overnight (>18 h) at 20 °C. After fixation, the cells were washed with PBS, centrifuged, resuspended in 0.25 mL of PBS with 5 μL of RNase (10 mg/mL, Sigma), and then incubated for 1 h at 37 °C. A total of 10 μL of propidium iodide (PI) (10 mg/mL, Sigma) was then added and incubated for 15 min in the dark. Stained cells were run in Guava Easycyte5 (Millipore) and analyzed using Guava InCyte software (Millipore). Apoptosis Study by Flow Cytometry. AGS cells were seeded on 12-well plates (1 × 105 cells/well) for 24 h, and treated as described. Floating and attached cells were both collected after the incubation

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ASSOCIATED CONTENT

S Supporting Information *

Effects of 1 on the viability of the cancer cells SW480, HCT116, and DLD1 and noncancer cells HEK293, the involvement of caspase in the antiproliferative activity of 1, Western blot analysis on the levels of GSK3β and CK1α and phosphorylated β-catenin in AGS cells after treatments with different concentrations of 1, effects of the z-VAD-fmk and calpastatin pretreatments on βcatenin levels in AGS cells treated with 1, TOP and FOP activities of 2, antiproliferative activity of 2 against AGS cells, and activity of 2 to overcome TRAIL-resistance. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81-43-226-2923. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by KAKENHI Grant Number 23102008 from MEXT, Grant Numbers 26305001 and 25870128 from JSPS, the Cosmetology Research Foundation, the Hamaguchi Foundation for the Advancement of Biochemistry, and the Uehara Memorial Foundation. We thank Prof. J. Nathans (John Hopkins University School of Medicine) for providing the STF/293 cells and Prof. R. T. Moon (University of Washington) for the SuperFOPflash plasmid.

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REFERENCES

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DOI: 10.1021/np500933v J. Nat. Prod. XXXX, XXX, XXX−XXX