A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin signaling pathway

Article abstract of DOI:10.1016/j.ejphar.2019.01.047

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

Full text of DOI:10.1016/j.ejphar.2019.01.047

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
www.elsevier.com/locate/ejphar
PII:
S0014-2999(19)30073-1
https://doi.org/10.1016/j.ejphar.2019.01.047
EJP72188
DOI:
Reference:
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
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A vanillin derivative suppresses the growth of HT29 cells through the Wnt/β-catenin
signaling pathway
1
3
1
1
1
2
Wantong Ma , Xue Li , Peng Song , Qianqian Zhang , Zhengrong Wu , Jianhui Wang ,
2
1
1
1
1
4,*
Xiaofeng Li , Ruixiang Xu , Wenbin Zhao , Yuheng Liu , Huanxiang Liu , Xiaojun Yao ,
3
,*
1,*
Xiaoliang Tang , Peng Chen
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
xjyao@must.edu.mo
tangxiaol@lzu.edu.cn
chenpeng@lzu.edu.cn
*
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
2
012, 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.,
2
4
018; Torre et al., 2016). According to data from the China National Cancer Center, there are
.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).
3

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.,
2
016). 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
1
further purification. All of the solvents used were of analytical reagent grade. H NMR and
1
3
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 HNO (50
3
ml) and H SO (100 ml) was added dropwise to a mixture of 1,10-phenanthroline (4.86 g, 27
2
4
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 CH Cl . At the same time, the aqueous solution was extracted
2
2
with fresh CH Cl . Organic phases were combined and dried with anhydrous Na SO . The
2
2
2
4
yellow product was recovered by solvent evaporation under vacuum and recrystallized from
ethanol.
4

Synthesis of 4-(1H-imidazo[4,5-f][1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711).
,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
1
(
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
4
plates at a density of 1×10 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
2
.4 Migration and invasion assays
.4.1 Transwell cell migration assay. Cells were seeded at a density of 1×10 cells/0.1 ml in
5
serum free DMEM-F12 or RPMI-1640 into the upper chambers (8-μm pore size; Corning).
5

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
5
HT29 and HCT116 cells were seeded at a density of 1×10 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,
6

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
3
3
. Results
.1 Structure identification
.1.1 Structural identification of 1, 10-phenanthroline-5,6-dione
7

1
Yield: 3.07 g (54.1%). H NMR (400 MHz, DMSO-d , δ ppm): 8.99 (dd, J = 4.7, 1.8 Hz, 2H),
6
13
8
.39 (dd, J = 7.8, 1.8 Hz, 2H), 7.67 (dd, J = 7.8, 4.7 Hz, 2H). C NMR (100 MHz, DMSO-d ,
6
δ ppm):178.30, 154.88, 152.83, 136.20, 129.61, 125.76. ESI mass spectrum m/z: Calcd for
+
C H N O 210.04, Found: 210.09 [M+H] .
12
6
2
2
3
.1.2 Structural identification of IPM711
1
Yield: 0.56 g (99.0%). H NMR (400 MHz, DMSO-d , δ ppm): 9.56 (s, 1H), 9.03 (dd, J =
6
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,
1
3
J = 8.2 Hz, 1H), 3.96 (s, 3H). C NMR (100 MHz, DMSO-d , δ ppm): 152.00, 149.08,
6
1
5
48.52, 148.03, 144.40, 143.86, 130.12, 123.71, 121.99, 120.21, 119.96, 116.37, 110.89,
6.34.
+
ESI mass spectrum m/z: Calculated for C H N O 342.11, Found: 343.1153 [M+H] .
2
0
14
4
2
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 IC of 13.5 ± 0.78 μm and 9.7 ± 0.36
5
0
μm doses (P<0.05), respectively. The IC values of IPM711 and 5-FU were 5.6 ± 0.33 μm
5
0
and 19.7 ± 3.02 μm in HCT116 cells (P<0.05), respectively. In addition, NCM460, a normal
colon cell line, reached IC values of 19.5 ± 0.40 μm and 24.7 ± 3.01 μm after treatment with
50
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
8

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
9

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
1
0

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.
1
604FKCA110), the Fundamental Research Funds for the Central Universities of China
1
1

(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.
2
017-4-122 and Grant No. 2018-4-59).
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Shaddox, K.F., Kim, S., Davies, S.R., Wang, S., Wang, P., Kinsinger, C.R., Rivers, R.C.,
Rodriguez, H., Townsend, R.R., Ellis, M.J., Carr, S.A., Tabb, D.L., Coffey, R.J., Slebos,
R.J., Liebler, D.C., 2014. Proteogenomic characterization of human colon and rectal cancer.
Nature 513, 382-387.
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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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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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