β-catenin signaling pathway

β-catenin signaling pathway

Author’s Accepted Manuscript A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway Wantong Ma, Xue Li,...

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Author’s Accepted Manuscript A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway Wantong Ma, Xue Li, Peng Song, Qianqian Zhang, Zhengrong Wu, Jianhui Wang, Xiaofeng Li, Ruixiang Xu, Wenbin Zhao, Yuheng Liu, Huanxiang Liu, Xiaojun Yao, Xiaoliang Tang, Peng Chen

PII: DOI: Reference:

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S0014-2999(19)30073-1 https://doi.org/10.1016/j.ejphar.2019.01.047 EJP72188

To appear in: European Journal of Pharmacology Received date: 29 October 2018 Revised date: 25 January 2019 Accepted date: 28 January 2019 Cite this article as: Wantong Ma, Xue Li, Peng Song, Qianqian Zhang, Zhengrong Wu, Jianhui Wang, Xiaofeng Li, Ruixiang Xu, Wenbin Zhao, Yuheng Liu, Huanxiang Liu, Xiaojun Yao, Xiaoliang Tang and Peng Chen, A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2019.01.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway Wantong Ma1, Xue Li3, Peng Song1, Qianqian Zhang1, Zhengrong Wu1, Jianhui Wang2, Xiaofeng Li2, Ruixiang Xu1, Wenbin Zhao1, Yuheng Liu1, Huanxiang Liu1, Xiaojun Yao4,*, Xiaoliang Tang3,*, Peng Chen1,* 1

School of Pharmacy, Lanzhou University, 199 Donggang West Road, Lanzhou, 730000, PR

China 2

Department of Pathology, School of Medicine, Yale University, 310 Cedar Street, New

Haven, 06510, USA 3

College of Chemistry and Chemical Engineering, Lanzhou University, 222 Tianshui South

Road, Lanzhou, 730000, PR China 4

Macau Institute for Applied Research in Medicine and Health, State Key Laboratory of

Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau, PR China

[email protected] [email protected] [email protected] *

Correspondence: Tel.: +86 931 8915686

Abstract Colorectal cancer (CRC) is a common malignancy and the leading cause of cancer death worldwide. According to previous studies, vanillin possesses pharmacological and anticancer activities. In this work, we have modified the structure of vanillin to obtain a vanillin derivative called 4-(1H-imidazo [4,5-f][1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711), which has improved anticancer activity. The present study is intended to explore the anti-colorectal cancer activity of IPM711 in HT29 and HCT116 cells. The results of this study suggest that IPM711 can inhibit the growth, invasion and migration of HT29 and HCT116 cells. Western blot and molecular docking showed that IPM711 could bind to a Wnt/β-catenin signaling receptor to inhibit cell growth, invasion and migration in HT29 cells. Based on these results, IPM711 is a promising anticancer drug candidate for human colorectal cancer therapy. Graphical Abstract:

Keywords: Colorectal cancer, Invasion, Migration, Vanillin derivative, Wnt/β-catenin. 2

1. Introduction Colorectal cancer (CRC) is a malignant tumor that is a serious threat to human health (Haraldsdottir et al., 2014). Despite recent advances in radiotherapy, chemotherapy and surgical techniques used in the treatment of colorectal cancer, the overall survival rate of CRC patients remains consistently low (Wang et al., 2015). According to statistics from 2012, there were almost 1.69 million new cases of CRC globally. As of 2018, CRC will be the fourth most fatal cancer worldwide after lung, stomach, and liver cancers (Siegel et al., 2018; Torre et al., 2016). According to data from the China National Cancer Center, there are 4.29 million new incidences of cancer and more than 2.81 million cancer-related deaths (Chen et al., 2016). Chemotherapy is an essential treatment modality commonly used to inhibit cancer relapse and prolong patient life. However, despite its distinct anticancer properties, the side effects and selectivity of chemotherapy limit the scope of its use. In recent years, targeted treatment strategies have been frequently mentioned in the field of drug design. Therefore, understanding how signaling pathways relevant to CRC can be regulated would greatly propel the development of novel CRC treatment strategies. Interestingly, growing evidence indicates that CRC is not a single, uniform disease type, but rather consists of a group of molecularly heterogeneous diseases characterized by a series of genomic and epigenomic alterations (Inamura, 2018; Zhang et al., 2014). In addition, data from The Cancer Genome Atlas (TCGA) suggests that the Wnt signaling pathway is activated in 93% of nonhypermutated CRC and 97% of hypermutated CRC (Li et al., 2012; Sebio et al., 2014; Voorneveld et al., 2015). Therefore, the Wnt/β-catenin signaling is an appropriate drug target with the potential for treatment of CRC. Natural products are tightly associated with the Wnt/β-catenin signal pathway (Catalani et al., 2016), including polyphenols such as green tea polyphenols, epigallocathechin-3-gallate, and resveratrol (Bai et al., 2017; Sarkar et al., 2010).

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Vanillin (3-Methoxy-4-hydroxybenzaldehyde) is a pharmacological component of the Chinese herb gastrodia. Nehal M found that vanillin has anti-tumor effects (Elsherbiny et al., 2016). However, the anticancer activity of vanillin is inferior to that of the broad-spectrum anti-tumor drug 5-fluorouracil (5-FU). Based on these factors, we modified the structure of vanillin and obtained a vanillin derivative called 4-(1H-imidazo [4,5-f][1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711). The present study is intended to explore the anti-colorectal cancer activity of IPM711 in HT29 and HCT116 cells and elucidate the molecular mechanism of its anticancer activity. 2. Materials and Methods 2.1 Synthesis of IPM711 All the materials for synthesis were purchased from commercial suppliers and used without further purification. All of the solvents used were of analytical reagent grade. 1H NMR and 13

C NMR spectra were recorded at JNM-ECS-400 MHz and referenced to the solvent signals.

Mass spectra were performed using a Bruker Micro TOF ESI-TOF mass spectrometry. All pH measurements were performed using a pH-10C digital pH meter. Synthesis of -5,6-1,10-phenanthroline dione. An ice-cold solution of concentrated HNO3 (50 ml) and H2SO4 (100 ml) was added dropwise to a mixture of 1,10-phenanthroline (4.86 g, 27 mmol) and NaBr (28.5 g, 27.5 mmol) in an ice bath. The mixture was then refluxed for 4 h. After the completion of the reaction, the light yellow solution was poured over 200 ml of ice and then neutralized by addition of a saturated solution of NaOH. The solids were filtered off and repeatedly washed with CH2Cl2. At the same time, the aqueous solution was extracted with fresh CH2Cl2. Organic phases were combined and dried with anhydrous Na2SO4. The yellow product was recovered by solvent evaporation under vacuum and recrystallized from ethanol.

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Synthesis of 4-(1H-imidazo[4,5-f][1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711). 1,10-phenanthroline-5,6-dione (346 mg, 1.64 mmol), 4-hydroxy-3-methoxybenzaldehyde (250 mg 1.64 mmol), ammonium acetate (1.073 g, 8.5 equivalent) and 9 ml glacial acetic acid were added to a round bottom flask. The mixture was refluxed at 85 °C for 8 h. After the completion of the reaction, the pH was adjusted to neutrality by addition of a saturated solution of NaOH. The yellow solids precipitated were filtered, washed with water and dried in vacuo. This is illustrated in Scheme 1 and Fig. 1. 2.2 Cell culture Human colon carcinoma HT29 cells were maintained at 37 °C in a humidified 5% CO2 incubator and cultured in DMEM-F12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The colorectal cell lines HCT116 and the normal cell lines NCM460 were cultured in RPMI 1640 medium with 10% FBS and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) at 37 °C and 5% CO2. 2.3 MTT assay for cell proliferation A methylthiazolyl blue tetrazolium (MTT) spectrophotometric dye assay was performed to determine cell proliferation. HT29, HCT116 and NCM460 cells were seeded in 96-well plates at a density of 1×104 cells/well. Following overnight incubation, the cells were treated with different concentrations of IPM711 for 72 h. Cells were incubated for 4 h in 15 μl MTT solution (5 mg/ml PBS, pH 7.2) at 37 °C, followed by addition of 150 μl of solubilizing buffer (10% SDS in 0.01 N HCl). Cell viability of treated cells was calculated according to the data of untreated control cells using the formula = 1× (sample Abs)/ (control). Abs is the absorbance value at 570 nm. 2.4 Migration and invasion assays 2.4.1 Transwell cell migration assay. Cells were seeded at a density of 1×105 cells/0.1 ml in serum free DMEM-F12 or RPMI-1640 into the upper chambers (8-μm pore size; Corning).

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Both cell lines included a control group and a drug treatment group (HT29 = 10 μm, HCT116 = 4 μm). Each lower chamber was filled with 600 μl of media with 20% FBS. After incubation for 24 h, HT29 and HCT116 cells were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 10 min (Paek et al., 2014). Migrating cells were visualized by microscopy at 400× magnification and quantified. All experiments were performed in triplicate at a minimum, and the average values are reported. 2.4.2 Transwell cell invasion assay HT29 and HCT116 cells were seeded at a density of 1×105 cells/0.1 ml in serum free DMEM-F12 or RPMI-1640 into the upper chambers, which were precoated with Solarbio Matrigel (8 μm pore size; Corning) (Cai et al., 2015). Both cell lines included a control group and drug treatment group (HT29 = 10 μm, HCT116 = 4 μm). Each lower chamber was filled with 600 μl of 20% FBS medium. After incubation for 48 h (Horng et al., 2016), HT29 and HCT116 cells embedded in the membranes were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 10 min. Migrating cells were visualized and counted by microscopy at 400 × magnification. All experiments were conducted in triplicate at a minimum, and the average values are reported. 2.5 Western blot The total cell proteins were extracted with RIPA lysis solution (Solarbio) containing 1% phenylmethylsulfonyl fluoride (PMSF). The protein concentration was detected by the Bradford assay. Following 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane and then incubated for 1 h in Tris-buffered saline and Tween (TBST) containing 5% nonfat milk. GAPDH was used as an internal reference. The membrane was subsequently incubated at 4 °C overnight with primary antibodies against β-catenin (1:1000; bioss), GSK-3β (1:1000,

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bioss), p-β-catenin (1:1000, bioss), C-Myc (1:1000, bioss), E-cadherin (1:1000, bioss) and GAPDH (1:5000, bioss). Membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature for 2 h, followed by autoradiography, imaging and recording. The films were scanned, and the image analysis software ImageJ was used to analyze the grayscale images. The relative ratio was calculated by comparison to the expression of the internal reference protein. All experiments were performed in triplicate at a minimum, and the average values are reported. 2.6 Docking Molecular docking calculations were carried out using Glide in Schrödinger suite 10.1. The crystallographic structures of FZD4-CRD in complex with palmitoleic acid were retrieved from the Protein Data Bank (PDB code: 5UWG). For the complex structure, the Protein Preparation Wizard module in Schrödinger 10.1 was used to remove all crystallographic water molecules, add missing side chains and hydrogen atoms, assign protonated states and partial charges with the OPLS2005 force field, and minimize the whole crystal structure until the root-mean-squared deviation (RMSD) of the displacement of the nonhydrogen atoms reached 0.3 Å. IPM711 was prepared by the Ligprep module in Schrödinger. 2.7 Statistical analysis All results are presented as mean ± SD from independent experiments. Data were analyzed with ImageJ and SPSS 19 software. Statistical differences were analyzed by performing Student’s t-test for independent samples. Differences between control and IPM711 treated groups were considered significant when P<0.05. 3. Results 3.1 Structure identification 3.1.1 Structural identification of 1, 10-phenanthroline-5,6-dione

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Yield: 3.07 g (54.1%). 1H NMR (400 MHz, DMSO-d6, δ ppm): 8.99 (dd, J = 4.7, 1.8 Hz, 2H), 8.39 (dd, J = 7.8, 1.8 Hz, 2H), 7.67 (dd, J = 7.8, 4.7 Hz, 2H). 13C NMR (100 MHz, DMSO-d6, δ ppm):178.30, 154.88, 152.83, 136.20, 129.61, 125.76. ESI mass spectrum m/z: Calcd for C12H6N2O2 210.04, Found: 210.09 [M+H] +. 3.1.2 Structural identification of IPM711 Yield: 0.56 g (99.0%). 1H NMR (400 MHz, DMSO-d6, δ ppm): 9.56 (s, 1H), 9.03 (dd, J = 4.2, 1.7 Hz, 2H), 8.92 (d, J = 8.2 Hz, 2H), 7.84 (m, 3H), 7.75 (dd, J = 8.2, 2.0 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 3.96 (s, 3H). 13C NMR (100 MHz, DMSO-d6, δ ppm): 152.00, 149.08, 148.52, 148.03, 144.40, 143.86, 130.12, 123.71, 121.99, 120.21, 119.96, 116.37, 110.89, 56.34. ESI mass spectrum m/z: Calculated for C20H14N4O2 342.11, Found: 343.1153 [M+H] +. 3.2. IPM711 suppresses the cell growth of HT29, HCT116 and NCM460 cells. Data are shown in Fig. 2 (A, B and C). After treatment with different concentrations of IPM711 and 5-FU for 72 h, HT29 reached an inhibitory IC50 of 13.5 ± 0.78 μm and 9.7 ± 0.36 μm doses (P<0.05), respectively. The IC50 values of IPM711 and 5-FU were 5.6 ± 0.33 μm and 19.7 ± 3.02 μm in HCT116 cells (P<0.05), respectively. In addition, NCM460, a normal colon cell line, reached IC50 values of 19.5 ± 0.40 μm and 24.7 ± 3.01 μm after treatment with IPM711 and 5-FU (P<0.05), respectively. The results showed that the inhibitory effect of IPM711 on HT29 cells was less than that of 5-FU, but its inhibitory effect on HCT116 cells was higher than that of 5-FU. Meanwhile, the IC50 value for NCM460 cells was significantly different compare with that of HT29 (13.5 ± 0.78 μm, P<0.01) and HCT116 (5.6 ± 0.33 μm, P<0.01) cells after treatment with the same concentration of IPM711. As is shown in Fig. 2 (E and F), following treatment with IPM711 for different lengths of time (HT29 = 10 μm, HCT116 = 5 μm), we found that the OD value of the 48 h treatment group was higher than that of the 0 h group. This finding indicated that both groups of cells were

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growing within 48 h. Therefore, the next step was to rule out the effect of cell proliferation on the experiment. 3.3. IPM711 inhibits the migration of HT29 and HCT116 cells in vitro Transwell cell invasion and migration assays were performed to investigate the effects of IPM711 on HT29 and HCT116 cell migration and invasion in vitro. The results in Fig. 3 show that following treatment with IPM711, the invasive and migratory capacity of HCT116 and HT29 cells were noticeably decreased compared with the control group. 3.4. IPM711 inhibits HT29 cell growth through the Wnt/β-catenin signaling pathway As shown in Fig. 4A, the expression of E-cadherin was increased after treatment with IPM711 in both cell lines (P<0.05). The protein expression of β-catenin had the opposite tendency in different cells lines after treatment (P<0.05). The expression of c-Myc, a protein associated with cancer, was decreased in HT29 cells. However, the expression of c-Myc did not change in HCT116 cells (P>0.05). GSK-3β protein expression was similar to that of β-catenin (P>0.05). In addition, the expression of the inactive phosphorylated form of β-catenin was increased in HT29 and HCT116 cells (P<0.05) (An et al., 2008). 3.5. IPM711 binds to the membrane receptor protein FZD Docking results revealed that IPM711 can stably bind to the active pocket of FZD4-CRD with a docking score of -9.154 and a binding free energy of -72.82 kcal/mol. Furthermore, the binding mode analyses of IPM711 to FZD4-CRD indicate that the pi-pi stacking, the hydrogen bond interactions, the van der Waals interactions, as well as the hydrophobic interactions between IPM711 and FZD4-CRD are responsible simultaneously for the strong binding affinities. The hydrophobic interactions between IPM711 and FZD4-CRD are most critical for the binding. The benzene and pyridine rings make extensive van der Waals and hydrophobic contacts with the nonpolar side chains of several residues in the active sites, including Phe82, Pro84, Leu85, Val131, Leu132, Phe135, and Phe137. Moreover, pi-pi

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stacking (benzene of IPM711 with residues Phe157, Phe137) and hydrogen bonds (hydroxyl group of IPM711 with the carbonyl oxygen of Val131) are formed to stabilize the IPM711 in the active site (Fig. 5). 4. Discussion A growing amount of evidence suggests that natural products used as dietary supplements can reduce the risk of cancer (Borralho et al., 2007). After treatment with IPM711, a derivative of vanillin, we found that the growth, invasion and migration of HT29 and HCT116 cells were inhibited. In addition, OD-time curves of the two cell lines suggested that IPM711 could inhibit the cell growth of HT29 and HCT116 cells during the treatment period. The Wnt/β-catenin signaling pathway is highly activated in colorectal cancer. β-catenin is a key nuclear translocation protein. In the absence of the Wnt signal, most of the β-catenin binds to E-cadherin and a backbone protein to maintain its stability. Meanwhile, a small portion of β-catenin is degraded by binding to the APC, GSK-3β and Axin protein complex, making it unable to enter the nucleus to activate the downstream oncogenes (Novellasdemunt et al., 2015). When the Wnt signal is active, the APC, GSK-3 β, and Axin protein complex becomes inactive and thus cannot phosphorylate β-catenin. The results of the Western blots showed that the expression levels of β-catenin and c-Myc proteins were downregulated in HT29 cells, while the expression levels of E-cadherin and p-β-catenin were increased. E-cadherin is a protein that plays an important role in cell-cell adhesion (Peifer et al., 1992). Its expression can enhance the adhesion between cancer cells and reduces the risk of cancer cell metastasis, which may be related to the function of IPM711 in inhibiting invasion of both cell lines. Moreover, the results of docking revealed that the ability of IPM711 to inhibit the proliferation of HT29 cells may be related to its

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binding of the membrane receptor FZD, since the combination of Wnt signaling factor with this receptor has been inhibited. Interestingly, the growth of the CRC cell line HCT116 was inhibited following the same treatment. However, the Western blot results demonstrated the increased expression of E-cadherin protein. This was associated with the inhibitory effect on the invasion of HCT116 by IPM711. In addition, the increased expression of p-β-catenin suggested that there was a reduction in active β-catenin in the cytoplasm. However, the reason that the expression of β-catenin in the control group was lower than drug group may be due to a β-catenin mutation in the HCT116 cells (Kaler et al., 2012). Although we have discovered that the newly synthesized vanillin derivatives could inhibit the growth of colorectal cancer cells, the mechanism by which IPM711 mediates its cytotoxic effects on HCT116 cells may be different in HT29 cells. Unfortunately, this study did not find the mechanism of IPM711 that affects the expression of related proteins in HCT116 cells. Therefore, the specific mechanism of action remains to be further studied. 5. Conclusions In this work, we explored the biological activity of IPM711 in CRC cells. These results confirmed that IPM711 has a significant inhibitory effect on growth of HT29 and HCT116 compare with the normal colorectal cells NCM460. In addition, IPM11 suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway. Although additional CRC cell lines are needed to confirm the therapeutic effect of IPM711, the data presented here indicates that the vanillin derivative IPM711 is a promising strategy for the treatment of colorectal cancer. Acknowledgments This work was supported by Technology Program of Gansu Province (Grant No. 1604FKCA110), the Fundamental Research Funds for the Central Universities of China

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(Grant No. lzujbky-2017-197 and Grant No. lzujbky-2017-110), the Project of Lanzhou City for Innovative and Entrepreneurial Talents (Grant No. 2017-RC-73) and Science and Technology Project of Lanzhou City (Grant No. 2015-3-93, Grant No. 2016-3-75, Grant No. 2017-4-122 and Grant No. 2018-4-59).

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Scheme 1. Synthesis of IPM711.

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Fig. 1. 1H NMR spectrum of IPM711 in DMSO-d6. Fig. 2. The effect of IPM711, 5-FU on the growth of human colorectal cancer cell lines. (A, B, C) The effect of IPM711, 5-FU at the concentration of 5, 10, 15, 20, 25, 30, 40 μm on HT29, NCM460 and HCT116 cells. (D) All cell lines were treated with the same concentration of IPM711. (E,F) The OD of cells treated with IPM711 in HT29 and HCT116 cell lines. The viability percentage is presented as the mean ± standard deviation. *P < 0.05; ** P < 0.01, *** P < 0.001, IPM711 vs. 5-FU. Fig. 3. (A, C) Characterization of migratory and invasive capacity of HT29 and HCT116 cells treated with IMP711. (B, D) The number of HT29 and HCT116 cells visualized and quantified by microscopy. * P < 0.05; ** P < 0.01, *** P < 0.001 compared with the control. Fig. 4. The effect of IPM711 on Wnt/β-catenin signaling in CRC cells. (A) IPM711 significantly affects the expression of Wnt/β-catenin signaling proteins in HT29 cells compared with DMSO. (B) Statistical analysis. Bars indicate ± standard errors. * P < 0.05; ** P < 0.01, *** P < 0.001 compared with the control. Fig. 5. Molecular docking analysis between IPM711 and Frizzled binding. (A) The best possible binding mode of IPM711 on the Frizzled active site (the key residues are displayed). (B) The corresponding interactions of the docked complex (IPM711 & Frizzled).

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