(E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol inhibits growth of colon tumors in mice

In our previous study, we found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal showed anti-cancer effect, but it showed lack of stability and drug likeness. We have prepared several (E)-2,4-bis(p-hydroxyphenyl)-2-butenal analogues by Heck reaction. We selected two compounds which showed significant inhibitory effect of colon cancer cell growth. Thus, we evaluated the anti-cancer effects and possible mechanisms of one compound (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol in vitro and in vivo. In this study, we found that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol induced apoptotic cell death in a dose dependent manner (0-15 μg/ml) through activation of Fas and death receptor (DR) 3 in HCT116 and SW480 colon cancer cell lines. Moreover, the combination treatment with (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol and nuclear factor κB (NF-κB) inhibitor, phenylarsine oxide (0.1 μM) or signal transducer and activator of transcription 3 (STAT3) inhibitor, Stattic (50 μM) increased the expression of Fas and DR3 more significantly. In addition, (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol suppressed the DNA binding activity of both STAT3 and NF-κB. Knock down of STAT3 or NF-κB p50 subunit by STAT3 small interfering RNA (siRNA) or p50 siRNA magnified (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol-induced inhibitory effect on colon cancer cell growth. Besides, the expression of Fas and DR3 was increased in STAT3 siRNA or p50 siRNA transfected cells. Moreover, docking model and pull-down assay showed that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol directly bound to STAT3 and NF-κB p50 subunit. Furthermore, (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol inhibited colon tumor growth in a dose dependent manner (2.5 mg/kg-5 mg/kg) in mice. Therefore, these findings indicated that (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol may be a promising anti-cancer agent for colon cancer with more advanced research.


INTRODUCTION
Colorectal cancer (CRC, also known as colon cancer, bowel cancer) ranks third among the leading causes of cancer-associated death after lung and prostate cancer for men and after lung and breast cancer for women [1]. On the other hand, colon cancer is also one of the most curable cancers if it is detected in early stage through regular colonoscopy [2]. Systemic chemotherapy plays an integral part in advanced colon cancer treatments, however, 50% of patients respond poorly or have disease progression due to resistance to chemotherapeutic agents [3]. As present treatments for colon cancer patients are not so sufficient, it is urgent to develop appropriate novel chemo-preventive compounds.
Apoptosis is the process of programmed cell death which has an important role in anti-cancer effects of chemotherapeutics [4]. Activated death receptors (DRs) induce apoptosis through caspase activation [5]. DRs are activated by binding to their ligands (interaction of DR1 with TNF; Fas with FasL; DR3 with TWEAK; DR4 and DR5 with TRAIL; Ligand of DR6 has not been exactly defined) [6,7]. Activation of death receptors induces activation of caspase-8, which leads to the activation of downstream caspases, including caspase-9 and caspase-3, as well as the translocation of Bax to mitochondria leading to apoptosis [8]. Increase of death receptor expression could enhance susceptibility of cancer cells toward chemotherapeutics [9].
Signal Transducer and Activator of Transcription 3 (STAT3) belongs to the STAT family of proteins, which are both signal transducers and transcription factors [10]. STAT3 is a key signal transduction protein that mediates signaling by many cytokines, hormones, growth factors, and oncoproteins [11]. Once theses ligands bind to the specific transmembrane STAT3 receptor, STAT3 becomes activated by tyrosine phosphorylation and dimerizes through reciprocal Src homology 2-phosphotyrosine binding, and the dimeric STAT3 translocates to the nucleus, where it binds to consensus STAT3 binding sequences within the promoter region of target genes and thereby activates their transcription [11]. Phosphorylation of STAT3 performs a vital function in cell growth, proliferation, survival, differentiation, apoptosis, metastasis and angiogenesis [14][15][16][17]. Constitutively activated STAT3 has been identified in many cancers including colon cancer [18]. Studies in the past few years have provide compelling evidence for the critical role of aberrant STAT3 in malignant transformation and tumorigenesis, thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design [10]. Selected natural inhibitors of the STAT3 signaling pathway are Betulinic acid, Butein, Caffeic acid, Capsaicin, Celastrol, Cucurbitacins, Curcumin, Diosgenin, Guggulsterone, Honokiol and so on [13]. As STAT3 is activated by dimerization and then binds DNA to perform its functions, specific inhibitors targeting the disruption of their protein-protein binding or DNA-binding activity, are more promising agents [13].
On the other hand, drugs aimed at multiple pathways can be more efficacious and less vulnerable to acquire resistance because the disease system is less able to compensate for the action of two or more drugs simultaneously, and this approach can be particularly beneficial in cancers because oncogenesis is known to be a multistep process [13,19]. NF-κB is constitutively activated in human colorectal carcinoma tissue and colon cancer cells [20]. NF-κB plays a crucial role in the suppression of apoptosis as well as in the induction of cell proliferation and inflammation, NF-κB is closely associated with cancer development [21]. NF-κB acts as a cell survival factor through its regulatory role in the expression of an array of apoptotic (caspase-3 and Bax), antiapoptotic (Bcl-2 and IAP family), and cell proliferation genes (cyclooxygenase-2 and cyclins) [22]. NF-κB and STAT3 are rapidly activated in response to various stimuli including stresses and cytokines, although they are activated by entirely different signaling mechanisms. Once activated, NF-κB and STAT3 control the expression of anti-apoptotic, pro-proliferative and immune response genes some of which overlap and require transcriptional cooperation between the two factors [23]. Therefore, inhibition of NF-κB and STAT3 by chemotherapeutics is intended as a potential strategy to eliminate cancerous cells through induction of apoptosis.

Effect of (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol on the expression of apoptosis regulatory proteins, as well as the DNA binding activity of NF-κB and STAT3 in colon tumor tissues
To examine the relationship between colon tumor growth and apoptosis regulatory proteins, we performed Western blotting assay. We found the expression of Fas, DR3, cleaved caspase-3, cleaved caspase-8 and Bax was increased while the expression of Bcl-2 was decreased in a dose dependent manner (2.5 mg/kg-5 mg/kg) ( Figure  7A). The nucleus expression of p-STAT3, p-IκBα, p50 and p65 was decreased in a dose dependent manner (2.5 mg/ kg-5 mg/kg) ( Figure 7B). We also found the DNA binding activities of NF-κB ( Figure 7C) and STAT3 ( Figure 7D) were decreased in a dose dependent manner (2.5 mg/kg-5 mg/kg).
In the present study, we found that (E)-4-(3-(3,5dimethoxyphenyl)allyl)-2-methoxyphenol treatment increased the expression of apoptotic proteins such as Bax, cleaved caspase-3, cleaved caspase-8 as well as the expression of death receptors like DR3 and Fas in a concentration dependent manner. However, the expression of anti-apoptotic protein Bcl-2 was decreased. Several anti-apoptotic proteins, such as Survivin and members of the Bcl family (Bcl-xl, Bcl-2 and Mcl-1) which are known to be crucial for tumor cell survival, are direct target genes of STAT3 and are down-regulated as a consequence of STAT3 inhibition [25]. Several studies have proposed

Figure 7: Effect of (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol on the expression of apoptosis regulatory proteins and the DNA binding activity of STAT3 and NF-κB in colon cancer tumors. A. Expression of apoptosis regulatory
proteins was determined by Western blot analysis with antibodies against Fas, DR3, capase-3, caspase-8, Bax, Bcl-2 and β-actin (internal control). B. Cytosol extracted proteins were used to determine the expression of IκBα, p-IκBα and β-actin (internal control). Nuclear extracted proteins were used to determine the expression of p50, p65, STAT3, p-STAT3 and Histone H1 (internal control) in colon cancer tumors. Each band is representative for three experiments. C. & D. Tumors were lysed with A buffer and C buffer. Nuclear extracts were incubated in binding reactions of ³²p-end-labeled oligo nucleotide containing the STAT3 or NF-κB sequence. The present EMSA results are representative for three experiments.

Chemicals
Heck reaction was used for the synthesis starting from phenyl halide moieties with substituents (2.0 mmol) and allylbenzene moieties with substituents (2.0 mmol). Phenyl halide (2.0 mmol) and allylbenzene (2.0 mmol) were added with triphenylphosphine (105 mg, 0.4 mmol), Pd(OAc) 2 (44.9 mg, 0.2 mmol), and tributylamine (451 mg, 1.9 mmol) in a 25 ml round bottom flask and the reaction mixture was stirred for 2 h at 45 o C under argon atmosphere. The product was purified by flash silica gel chromatography using hexane and ethyl acetate (3:1 mixture v/v) as the mobile phase.

Cell culture
The HCT116, SW480 colon cancer cell lines and CCD-18Co colon epithelial normal cell line were obtained from American Type Culture Collection (Manassas, VA, USA). HCT116 was cultured in DMEM (Gibco, Life Technologies, Grand Island, NY) medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and 100 units/ml penicillin, 100 μg/ml streptomycin. SW480 was cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS and 100 units/ml penicillin, 100 μg/ml streptomycin. CCD-18Co was cultured in DMEM medium supplemented with 10% heat inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.1 mM non-essential amino acids. Cell cultures were then maintained in an incubator within a humidified atmosphere of 5% CO 2 at 37 o C.

Apoptosis evaluation
Colon cancer cells HCT116 and SW480 were cultured on 8-chamber slides for 24 h and then were treated with (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2methoxyphenol (0-15 μg/ml) for 24 h. TUNEL assays were performed by using the DeadEnd™ Fluorometric TUNEL System (Promega Corporation, Madison, USA) according to manufacturer's instructions. Total number of cells in a given area was determined by using DAPI (Vector Laboratories, Inc., Burlingame, CA) staining. The cells were then observed through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany). The apoptotic index was determined as the number of TUNEL-positive stained cells divided by the total cell number counted x100%.

Electrophoretic mobility shift assay
The DNA binding activity of STAT3 was determined using EMSA according to the manufacturer's recommendations (Promega). In short, HCT116 and SW480 cells were cultured on 100-mm culture dishes. After treatment with (E)-4-(3-(3,5-dimethoxyphenyl) allyl)-2-methoxyphenol for 2 h, the cells were washed twice with PBS, followed by the addition of 1 ml of phosphate buffered saline (PBS), and the cells were scraped into a cold eppendorf tube. The cells were lysed in ice-cold buffer A (10 mM HEPES, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 0.1% protase inhibitor, 0.1% phosphatase inhibitor and 0.5% NP40) for 30 minutes, and centrifuged for 6 minutes at 6,000 rpm. The residual pellet was resuspended in buffer C (10 mM HEPES, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.2 mM PMSF, 0.1% protase inhibitor, 0.1% phosphatase inhibitor, 420 mM NaCl, 0.2 mM EDTA and 20% glycerol). After incubation at 4 o C for 1 h, the lysate was centrifuged for 15 minutes at 13,000 rpm and then nuclear extracts were prepared and processed for EMSA as previously described. The relative densities of the DNA-protein binding bands were scanned by densitometry using MyImage (SLB), and quantified by Labworks 4.0 software (UVP, Inc., Upland, CA).

Transfection of siRNA
Colon cancer cells were plated in 6-well plates (2 x 10 5 cells / well) and were transiently transfected with siRNA, using a mixture of siRNA and the WellFect-EX PLUS reagent in OPTI-MEM, according to the manufacturer's specification (WelGENE, Seoul, Korea). The transfected cells were treated with (E)-4-(3-(3,5dimethoxyphenyl)allyl)-2-methoxyphenol (10 μg/ml) for 24 h and then used for detecting cell viability and protein expression.

Docking experiment
Docking studies between STAT3 or p50 and (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol was performed using Autodock VINA [44]. Only one monomer of the homo-dimeric STAT3 or p50 crystal structure was used in the docking experiment and conditioned using AutodockTools by adding all polar hydrogen atoms. Three dimensional structures of STAT3-DNA complexes and p50-DNA complexes were retrieved from the Protein Data Bank (PDB codes: STAT3-3CWG, p50-1VKX). Starting from the co-crystallized complexes, the STAT3 or p50 monomer chain, (E)-4-(3-(3,5-dimethoxyphenyl) allyl)-2-methoxyphenol for docking were prepared using Maestro graphical interface. The grid box was centered on the STAT3 or p50 monomer and the size of the grid box was adjusted to include the whole monomer. Docking experiments were performed at various exhaustiveness values of the defaults: 16, 24, 32, 40 and 60. Molecular graphics for the best binding model was generated using Discovery Studio Visualizer 2.0.

Antitumor activity study in in vivo xenograft animal model
Eight-week-old male BALB/C nude mice were purchased from Orient-Bio (Gyunggi-do, Korea). The mice were maintained in accordance with the Korea Food and Drug Administration guidelines as well as the regulations for the care and use of laboratory animals of the animal ethics committee of Chungbuk National www.impactjournals.com/oncotarget University (CBNU-278-11-01). Human colon cancer cell line HCT116 cells were injected subcutaneous (1 x 10 7 cells/0.1 ml PBS/animals) with a 27-gauge needle into the right lower flanks in carrier mice. After 14 days, when the tumors had reached an average volume of 100-150 mm 3 , the tumor-bearing nude mice were intraperitoneally (i.p.) injected with (E)-4-(3-(3,5-dimethoxyphenyl)allyl)-2-methoxyphenol (2.5 mg/kg and 5 mg/kg dissolved in 0.01% DMSO) twice per week for 3 weeks. The group treated with 0.01% DMSO was designed as the control. The weight and tumor volume of the animals were monitored twice per week. The tumor volumes were measured with vernier calipers and calculated by the following formula: (A x B 2 )/2, where A is the larger and B is the smaller of the two dimensions. At the end of the experiment, the animals were sacrificed. The tumors were separated from the surrounding muscles and dermis, excised and weighed.

Immunohistochemistry
The animal tissues were fixed in 4% paraformaldehyde and cut into 10 μm sections using a freezing microtome (Thermo Scientific, Germany). The sections were stained with hematoxylin and eosin (H&E) for pathological examination. For immunohistological staining, tumor sections were incubated with primary antibody against PCNA, Fas, DR3, active caspase-3, phospho-STAT3, p50 (1:500, Abcam, Cambridge, UK). After rinse in phosphate buffered saline (PBS), the sections were subject to incubation in biotinylated secondary antibody. The tissue was incubated for 1 h in an avidin-peroxidase complex (ABC, Vector Laboratories, Inc., Burlingame, CA). After washing in PBS, the immunocomplex was visualized using 3, 3-diaminobenzidine solution (2 mg/10 ml) containing 0.08% hydrogen peroxide in PBS. Sections were dehydrated in a series of graded alcohols, cleared in xylene and coverslipped using Permount (Fisher Scientific, Suwanee, GA).

Statistical analysis
The data was analyzed by GraphPad Prism 4 software (Version 4.03, GraphPad Software, La Jolla, CA). Data was presented as mean ± S.D. The differences in all data were assessed by one-way analysis of variance. When the p value in the ANOVA test indicated statistical significance, the differences were assessed by the Dunnet's test. A value of p < 0.05 was considered to be statistically significant.