Plumbagin Downregulates Wnt Signaling Independent of p53 in

May 14, 2014 - Plumbagin (1), a naphthoquinone, induces cell death and affects various signaling pathways in cancer cells. Wnt signaling is active ...
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Plumbagin Downregulates Wnt Signaling Independent of p53 in Human Colorectal Cancer Cells Dinesh Raghu and Devarajan Karunagaran* Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India 600036

ABSTRACT: Plumbagin (1), a naphthoquinone, induces cell death and affects various signaling pathways in cancer cells. Wnt signaling is active constitutively in colorectal cancer and plays an important role in its progression and pathogenesis. It was hypothesized that 1 is likely to modulate Wnt signaling, and this compound was studied for its effect on this pathway in human colorectal cancer cells. Plumbagin (1) was found to downregulate Wnt signaling when assessed by a TOPFlash/FOPFlash reporter activity assay and also decreased the expression of several coactivators and downstream targets of Wnt signaling such as β-catenin, TCF7L2, p300, Bcl9l, c-Myc, vimentin, and cyclinD1 in SW620 colorectal cancer cells. Using isogenic HCT116p53+/ + and HCT116p53−/− colorectal cancer cells, it was found that compound 1-mediated downregulation of Wnt signaling is p53independent. Interestingly, treatment with 1 upregulated the expression of HBP1 (a negative regulator of Wnt signaling) in these cells. The results obtained show for the first time that downregulation of Wnt signaling could be one of the molecular mechanisms by which plumbagin exerts its inhibitory effects in human colorectal cancer cells.

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of cyclin D1,13 and modulates the acetyltransferase activity of p300.14 On the basis of such evidence, it was hypothesized that 1 may downregulate Wnt signaling, and it was decided to study its effects on Wnt signaling in colorectal cancer cells. Herein, it is reported for the first time that 1 indeed downregulates Wnt signaling in human colorectal cancer cells and that this effect is independent of p53 and may involve an increased expression of HBP1, a known negative regulator of Wnt signaling.

nt signaling is important in early development during axis formation, cell fate specification, cell polarity determination, and cell migration.1 It is also essential for maintaining homeostasis by self-renewal of intestinal epithelial cells,2 and deregulation in the Wnt signaling pathway can lead to cancer.3 Wnt signaling is initiated with the interaction of its ligand(s) with a frizzled receptor and activation of β-catenin, for which the stability is regulated by the destruction complex containing axin, GSK-3β, and APC. In the absence of Wnt ligand, casein kinase I (CKI) and glycogen synthase kinase-3β (GSK-3β) phosphorylate β-catenin, leading to recruitment of βTrCP-containing E3 ubiquitin ligase, for proteasomal degradation. During Wnt signaling, the destruction complex is inactivated by dishevelled protein, and free β-catenin regulates the transcription of several genes.3 In colorectal cancer, Wnt signaling is constitutively activated due to an APC mutation, a point mutation at the GSK-3β phosphorylation site at the Nterminal site of β-catenin, leading to stabilized β-catenin and promoting tumorigenesis.4 Plumbagin (1), a naphthoquinone constituent of Plumbago zeylanica L. (Plumbaginaceae), has been used in traditional medicine as an antifungal, antibacterial, and anti-inflammatory agent.5 Compound 1 is known to exhibit proapoptotic,6 antiangiogenic,7 and antimetastatic effects in cancer cells.8 Plumbagin is also known to inhibit NF-κB,9 JNK,10 PKCε, and STAT-3.11 This compound activates GSK-3β by inhibiting its inhibitory phosphorylation at Ser9,12 decreases the expression © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Plumbagin (1) Decreases the Viability of SW480 and SW620 Cells in a Concentration-Dependent Manner. Viabilities of SW480 and SW620 cells were observed on plumbagin (1) treatment at various concentrations for 24 or 48 h using an MTT assay, and the IC50 values (inhibitory concentrations 50%) for 1 were determined. Compound 1 decreased the viabilities of SW480 and SW620 cells, and the Received: December 4, 2013 Published: May 14, 2014 1130

dx.doi.org/10.1021/np4010085 | J. Nat. Prod. 2014, 77, 1130−1134

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Figure 1. Effects of plumbagin (1) on the viability of SW480 and SW620 cells (A and B, respectively). Cells were treated with 1 (0−20 μM) for 24 or 48 h, and cell survival was assessed with the MTT assay. Data are expressed as the means ± SE from three independent experiments.

IC50 values were found to be 12 and 7.3 μM for SW480 cells and 7.7 and 7.4 μM for SW620 cells at 24 and 48 h, respectively (Figure 1A,B). These results suggest that 1 decreases the viability of colorectal cancer cells in a concentration-dependent manner and that SW620 cells were more sensitive to 1 than SW480 cells at 24 h (Figure 1A,B). Hence, SW620 cells were used for further experiments. Plumbagin (1) Downregulates the Transcriptional Activity of TCF/LEF in Human Colorectal Cancer Cells. Since Wnt signaling plays an important role in colorectal cancer progression and pathogenesis,15 it was of interest to check the effect of 1 on Wnt signaling. During Wnt signaling, β-catenin translocates to the nucleus and interacts with T-cell factor (TCF)/lymphoid enhancer factor (LEF) in the nucleus and thereby controls the downstream gene expression by binding to a specific DNA sequence.16 TOPFlash plasmid has TCF/LEF binding domains, and the FOPFlash plasmid contains mutant TCF/LEF binding domains,17 so these were used to assess the effects of 1 on Wnt signaling. Compound 1 downregulated the luciferase activity of TOPFlash but not that of FOPFlash in a concentration-dependent manner in SW620 cells (Figure 2) and SW480 cells (data not shown). These data showed that 1 reduces the transcriptional activity of TCF/LEF significantly in colorectal cancer cells at concentrations (1−3 μM) lower than its IC50 values, suggesting the downregulation of Wnt signaling by 1. Plumbagin (1) Decreases the Expression of β-Catenin and Other Coactivators of Wnt Signaling. Many coactivators of Wnt signaling such as β-catenin,4 p300,18 TCF7L2,19 and Bcl9l20 are deregulated in colorectal cancer cells and aid in cancer progression. To study the effect of 1 on the expression of these coactivators, SW620 cells were treated with plumbagin, and their expression levels were quantified by Western blotting. Upon treatment with 1, the protein levels of

Figure 2. Effect of plumbagin (1) on the transcriptional activation of TCF/LEF promoter. SW620 cells were treated with DMSO (control) or 1 (1−3 μM) for 24 h, and TOPFlash/FOPFlash luciferase activity was carried out as described in the Experimental Section. Luciferase activity was normalized to Renilla luciferase activity. Data are expressed as the means ± SE from three independent experiments and considered significant if p values were found to be less than 0.05.

β-catenin, p300, TCF7L2, and Bcl9l were reduced when compared with those from the DMSO control (Figure 3), and similar results were obtained in SW480 cells (data not shown). These results suggest that 1 downregulates the expression of βcatenin and that of other coactivators of Wnt signaling, p300, TCF7L2, and Bcl9l, in colorectal cancer cells. Expression of Downstream Targets of Wnt Signaling Is Lower in Plumbagin (1)-Treated Colorectal Cancer Cells. To investigate the consequences of downregulation of Wnt signaling by compound 1, the expression levels were analyzed for the downstream targets (c-Myc, cyclinD1, and vimentin) of Wnt signaling on treatment with 1. The expression levels of c-Myc, cyclinD1, and vimentin were reduced to ca. 50% in 1-treated SW620 cells when compared to 1131

dx.doi.org/10.1021/np4010085 | J. Nat. Prod. 2014, 77, 1130−1134

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Figure 3. Effect of plumbagin (1) on the expression of coactivators of Wnt signaling. SW620 cells were treated with DMSO (control) or 6 μM 1 for 24 h, and the expression levels of β-catenin, TCF7L2, Bcl9l, p300, or β-actin (internal control) were analyzed by Western blotting using the respective antibodies. Similar results were obtained in three independent experiments.

treatment with this naphthoquinone upregulated HBP1 protein levels in SW620 cells (1.68-fold), HCT116 p53+/+ cells, and HCT116 p53−/− cells compared to their corresponding DMSO controls (Figure 6). These results showed that 1 increases the expression of HBP1 in colorectal cancer cells, and this may account for the downregulation of Wnt signaling by 1. In agreement with the results showing that plumbagin (1) decreases the viability of SW480, SW620, HCT116 p53+/+, and HCT116 p53−/− colorectal cancer cells, this compound has been shown to inhibit the viability of HT29 and HCT15 colorectal cancer cells.23 The Wnt pathway is the major deregulated pathway that aids in colorectal cancer progression with APC mutation24 and mutation at the GSK-3β phosphorylation site of β-catenin, leading to the formation of free and stable β-catenin, which can trigger malignancies.4,17 Data showing that plumbagin downregulated the stabilized β-catenin in SW620 cells (with an APC mutation) and HCT116 colorectal cancer cells (with a point mutation at the GSK-3β phosphorylation site in β-catenin) suggest that downregulation of Wnt signaling may be an important mechanism by which 1 acts. Since β-catenin modulates transcriptional activities through T-cell factor/lymphoid enhancer factor,25 plumbagin modulated the downregulation of transcriptional activity and the expression level of TCF7L2 in colorectal cancer cells to support this observation. In agreement with this, 1 also downregulated the expression of Bcl9l, which modulates translocation and transcriptional activity of β-catenin.20 Compound 1 has been shown to decrease the lysine acetyltransferase activity of p300,14 which coactivates the transcriptional activity of TCF7L2 by acetylating β-catenin and increases TCF7L2−β-catenin binding.18 Given that 1 treatment decreased the expression of cyclinD1, c-Myc, and vimentin, the downstream targets of Wnt signaling, the presently obtained data confirmed that 1 can effectively downregulate Wnt signaling in human colorectal cancer cells. Curcumin downregulates Wnt signaling in hepatocellular carcinoma cells, resulting in suppression of cyclinD1 and cMyc.26 Although 1 is known to increase the stability of p53 in A549 cells,10 the current data using HCT116 p53+/+ cells and HCT116 p53−/− cells suggest that the mechanism of inhibition of Wnt signaling is p53-independent. An increase mediated by 1 in the expression of HBP1, an HMG box repressor that inhibits Wnt signaling by competing for TCF/ LEF binding to DNA,22 is likely to be a potential mechanism by which plumbagin downregulates Wnt signaling. Epigallocatechin 3-O-gallate, a phenol from green tea, downregulates Wnt signaling through HBP1.27 Curcumin is known to downregulate Wnt signaling in cancer cells, leading to apoptosis and inhibition of proliferation26 and migration.28 Downregulation of Wnt signaling by plumbagin could be one of the mechanisms by which it induces apoptosis and inhibits proliferation29 and migration,30 although this remains to be investigated.

the DMSO control, thereby confirming the downregulation of Wnt signaling induced by 1 (Figure 4).

Figure 4. Effect of plumbagin (1) on the expression of downstream targets of Wnt signaling. SW620 cells were treated with DMSO (control) or 6 μM 1 for 24 h, and the expression levels of vimentin, cyclin D1, c-Myc, or β-actin (internal control) were analyzed by Western blotting using the respective antibodies. Similar results were obtained in three independent experiments.

Plumbagin (1) Downregulates Wnt Signaling through a p53-Independent Mechanism. As p53 is known to downregulate Wnt signaling,21 it was of interest to understand the role of p53 (if any) in 1-mediated downregulation of Wnt signaling. To this end, an isogenic cell system, HCT116 p53+/ + and HCT116 p53−/− colorectal cancer cells, with constitutively active Wnt signaling, was used. The absence of p53 protein expression was confirmed initially in HCT116 p53−/− cells, and its presence in HCT116 p53+/+ cells demonstrated by Western blotting, whereas β-actin (control protein) could be detected in both cell lines used (Figure 5A). The IC50 (20 μM) values for 1 were similar in both HCT116 p53+/+ and HCT116 p53−/− colorectal cancer cells (Figure 5B,C), and it also downregulated TOPFlash/FOPFlash luciferase activity equally well in both cell lines at two different concentrations (Figure 5D,E). Next, the effect of 1 was checked on β-catenin, and it was found that the protein levels of βcatenin decreased by 33% in HCT116 p53+/+ cells and 20% in HCT116 p53−/− cells on treatment with 1 when compared to their corresponding DMSO controls (Figure 5F). These results show that 1 downregulated Wnt signaling in a p53-independent manner. Plumbagin (1) Induces the Expression of HBP1, a Negative Regulator of Wnt Signaling. HBP1 negatively regulates Wnt signaling by interacting with TCF7L2 (TCF4) and preventing its DNA binding.22 Hence, the effect of 1 was investigated on the expression of HBP1, and it was found that 1132

dx.doi.org/10.1021/np4010085 | J. Nat. Prod. 2014, 77, 1130−1134

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Figure 5. Effect of plumbagin (1) on viability and Wnt signaling in HCT116 p53+/+ and HCT116 p53−/− colorectal cancer cells. (A) Expression of p53 in HCT116 p53+/+ and HCT116 p53−/− colorectal cancer cells was analyzed by Western blotting. (B) HCT116 p53+/+ and (C) HCT116 p53−/− cells were treated with 1 (0−30 μM) for 24 h, and cell survival was assessed using the MTT assay. (D) HCT116 p53+/+ and (E) HCT116 p53−/− cells were treated with DMSO (control) or 1 (5 and 10 μM) for 24 h, and TOPFlash/FOPFlash luciferase activity was carried out as described in the Experimental Section. Luciferase activity was normalized to Renilla luciferase activity. Data are expressed as the means ± SE from three independent experiments and considered significant if p values were found to be less than 0.05. (F) HCT116 p53+/+ and HCT116 p53−/− cells were treated with DMSO (control) or 10 μM 1 for 24 h, and the expression levels of β-catenin or β-actin (internal control) were analyzed by Western blotting using the respective antibodies. Similar results were obtained in three independent experiments. monolayers in a tissue culture flask fed with complete DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) containing 100 mg/L penicillin and 66.6 mg/L streptomycin. Cells were incubated in a humidified incubator at 37 °C with 5% CO2. During the experiment, the cells were washed using 1× PBS−EDTA, trypsinized using 0.25% trypsin, neutralized with complete DMEM, and centrifuged at 1500 rpm for 5 min, and the supernatant was aspirated, whereas the pellet was suspended in complete DMEM for seeding or culturing. Cytotoxicity Assay. Cell viability was assessed using MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye. Briefly, 5000 cells/well were seeded in 96-well plates and allowed to adhere overnight. Cells were treated with different concentrations of plumbagin (1) for 24 or 48 h, and MTT (5 mg/mL) was added and incubated for 3.5 h, followed by aspiration of the medium and solubilization of the formazan crystals by adding DMSO. After 5 min, the color developed was read as optical density (OD) using a Bio-Rad 680 model colorimeter at 570 nm with 655 nm as background reference. Luciferase Assay. Cells (3 × 104/well) were seeded in a 96-well plate, and TOPFlash/FOPFlash or Renilla plasmids (200 ng) were transfected with lipofectin (Invitrogen), using basal medium (DMEM without antibiotics and FBS). After 6 h of transfection, the basal medium was aspirated followed by plumbagin (1) treatment using complete DMEM at specified concentrations. The cells were lysed utilizing lysis buffer containing potassium phosphate buffer (1 M K2HPO4 and 1 M KH2PO4), 0.01% TritonX-100, and 100 mM dithiothreitol for 20 min. The dual luciferase assay was performed with luciferase assay buffer containing 25 mM glycylglycine, 15 mM phosphate buffer at pH 8.0, 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol, 15 mM magnesium sulfate, 75 μM luciferin, and 6355 μL of water for 7 mL of buffer and Renilla assay buffer containing 1.1 M NaCl, 2.2 mM disodium EDTA, 0.72 M KH2PO4, 0.44 mg/mL bovine serum albumin (BSA), 1.3 mM sodium azide,1.43 μM coelenterazine, and 3400 μL of water for 7 mL of buffer. Luciferase

Figure 6. Effect of plumbagin (1) on expression of HBP1, a negative regulator of Wnt signaling. SW620, HCT116 p53+/+, and HCT116 p53−/− cells were treated with DMSO (control) or 6 or 10 μM 1 for 24 h, and the expression levels of HBP1 or β-actin (internal control) were analyzed by Western blotting using the respective antibodies. Similar results were obtained in three independent experiments.

In conclusion, the results of this investigation demonstrated that plumbagin (1) inhibits Wnt signaling by transcriptional regulation of the TCF/LEF promoter, which may be due to an increase in the expression of HBP1 with a concomitant decrease in the expression of β-catenin, p300, TCF7L2, and Bcl9l, leading to the downregulation of cyclinD1, c-Myc, and vimentin. It was found also that the effect of 1 on Wnt signaling is p53-independent. The data obtained have thus unraveled a novel mechanism by which 1 acts, warranting further analysis on the functional consequences of the downregulation of Wnt signaling by plumbagin.

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EXPERIMENTAL SECTION

Chemicals. Plumbagin (1) (purity >98%) and MTT reagent were purchased from Sigma (St. Louis, MO, USA), and a stock solution of each was prepared by dissolving an appropriate quantity in dimethyl sulfoxide (DMSO) of cell-culture grade from Sigma. Cell Culture. Human colorectal cancer cells (SW480 and SW620 from ATCC, Manassas, VA, USA; HCT116p53+/+ and HCT116p53−/− were gifts from Dr. Bert Vogelstein, Johns Hopkins University, Baltimore, MD, USA) were maintained as adherent 1133

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assay reading was measured by photon counting using a Spectramax L instrument (Molecular Devices). Western Blotting. Cells (106/well) were seeded in six-well plates and treated with 6−10 μM plumbagin (1) and DMSO as a control for 24 h, after which the cells were collected and lysed in a lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM β-glycerophosphate, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 0.5% sodium deoxycholate, 1 mM PMSF, 20 mM NaF, 1% protease inhibitor) for 1 h on ice with intermittent vortexing. The lysates were centrifuged at 12 000 rpm for 10 min at 4 °C, and the supernatants collected were estimated using a Bradford assay. Equivalent amounts of protein (50− 75 μg) were resolved by 8−10% SDS-PAGE and transferred onto a PVDF membrane (Amersham). After blocking with Tris buffer saline (TBS) [500 mM NaCl, 20 mM Tris-HCl (pH 7.4)] containing 5% skimmed milk or 5% bovine serum albumin for 1 h, the membranes were incubated overnight at 4 °C with specific primary antibodies (1:1000). The membranes were subjected to three washes of 10 min each in TBS containing 0.1% Tween 20 (Sigma) and further incubated with HRP-conjugated secondary antibodies (1:10 000) (Jackson) at room temperature for 1 h, and proteins were detected by chemiluminescence (Versadoc, Bio-Rad) with an Enhanced Chemiluminescence kit (Amersham). Statistical Analysis. Values are expressed as the means ± SE of at least three independent experiments. Differences between control and treated samples were analyzed using a Student t-test, with p values of