NO2 functionalized coumarin derivatives suppress cancer progression and facilitate apoptotic cell death in KRAS mutant colon cancer

Article abstract of DOI:10.1016/j.cbi.2019.06.021

Colon cancer is one of the most lethal cancers worldwide even with the significant progress made in screening techniques and therapeutic agents. Genetic mutations in tumors complicated the treatments, and the survival rate remains low for patients at late or metastatic stages. KRAS gene mutation which leads to failure of the EGFR targeted therapies stands for an example of the challenges in clinical sites. Therefore, development of novel agents for colon cancer treatment is in need. Natural and synthetic coumarin derivatives have been suggested with various biological activities with pharmacologic potential including anti-cancer capacity. Here in this study, five coumarin derivatives, include trifluoromethyl-, dimethoxy-, and/or nitro-substitutions at different positions, were synthesized. Their cancer inhibition potential was investigated in various cancer cell lines. Our data demonstrated that one nitro-coumarin derivate, 5,7-Dimethoxy-4-methyl-6-nitro-chromen-2-one, exhibits cytotoxicity specifically towards colon cancer cells under competitive EC₅₀. Our results showed that this compound can effectively suppress colon cancer cells harboring either wild type or mutant KRAS genes, and that it could inhibit short-term proliferation, long term proliferation, and migration capacities of cancer cells. Finally, we demonstrated that this coumarin derivate facilitates cancer cell death through activation of apoptosis pathway. Our results suggest that this coumarin derivate is a promising lead drug worth further investigation and development for future cancer treatment.

Full text of DOI:10.1016/j.cbi.2019.06.021

Accepted Manuscript
NO functionalized coumarin derivatives suppress cancer progression and facilitate
2
apoptotic cell death in KRAS mutant colon cancer
Mei-Hsiang Lin, Jou-Shan Wang, Yi-Chen Hsieh, Jia-Huei Zheng, Er-Chieh Cho
PII:
S0009-2797(19)30315-1
https://doi.org/10.1016/j.cbi.2019.06.021
CBI 8708
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 6 March 2019
Revised Date: 17 May 2019
Accepted Date: 10 June 2019
Please cite this article as: M.-H. Lin, J.-S. Wang, Y.-C. Hsieh, J.-H. Zheng, E.-C. Cho, NO
2
functionalized coumarin derivatives suppress cancer progression and facilitate apoptotic cell death
in KRAS mutant colon cancer, Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/
j.cbi.2019.06.021.
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ACCEPTED MANUSCRIPT
NO2 functionalized coumarin derivatives suppress cancer progression and
facilitate apoptotic cell death in KRAS mutant colon cancer
Mei-Hsiang Lin1, Jou-Shan Wang1, Yi-Chen Hsieh2,3, Jia-Huei Zheng1, Er-Chieh Cho1,4, *
Mei-Hsiang Lin. 1School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei,
Taiwan. <mhl00001@tmu.edu.tw>
1
Jou-Shan Wang. School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei,
Taiwan. <staphanie22@gmail.com>
Yi-Chen Hsieh.2PhD Program for Neural Regenerative Medicine, College of Medical Science and
Technology, Taipei Medical University, Taipei, Taiwan; 3PhD Program in Biotechnology Research and
Development, College of Pharmacy, Taipei Medical University, Taiwan. <ychsieh@tmu.edu.tw>
Jia-hui Zheng. 1School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
<wendy31400@hotmail.com>
Er-Chieh Cho. 1School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
4Master Program in Clinical Pharmacogenomics and Pharmacoproteomics, College of Pharmacy,
Taipei Medical University, Taiwan. <echo@tmu.edu.tw>
*Corresponding author: Dr. Er-Chieh Cho: School of Pharmacy, College of Pharmacy, Taipei
Medical University, 250 Wuxing Street, Taipei 110, Taiwan. echo@tmu.edu.tw; Fax:
+886-2-2739-0671.
Keywords
Coumarin derivatives; apoptosis; cancer inhibition; KRAS mutant colon cancer.

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Abstract
Colon cancer is one of the most lethal cancers worldwide even with the significant
progress made in screening techniques and therapeutic agents. Genetic mutations in
tumors complicated the treatments, and the survival rate remains low for patients at
late or metastatic stages. KRAS gene mutation which leads to failure of the EGFR
targeted therapies stands for an example of the challenges in clinical sites. Therefore,
development of novel agents for colon cancer treatment is in need. Natural and
synthetic coumarin derivatives have been suggested with various biological activities
with pharmacologic potential including anti-cancer capacity. Here in this study, five
coumarin derivatives, include trifluoromethyl-, dimethoxy-, and/or nitro- substitutions
at different positions, were synthesized. Their cancer inhibition potential was
investigated in various cancer cell lines. Our data demonstrated that one
nitro-coumarin derivate, 5,7-Dimethoxy-4-methyl-6-nitro-chromen-2-one, exhibits
cytotoxicity specifically towards colon cancer cells under competitive EC50. Our
results showed that this compound can effectively suppress colon cancer cells
harboring either wild type or mutant KRAS genes, and that it could inhibit short-term
proliferation, long term proliferation, and migration capacities of cancer cells. Finally,
we demonstrated that this coumarin derivate facilitates cancer cell death through
activation of apoptosis pathway. Our results suggest that this coumarin derivate is a
promising lead drug worth further investigation and development for future cancer
treatment.
Keywords
Coumarin derivatives; apoptosis; cancer inhibition; KRAS mutant colon cancer.
Abbreviations
EGFR: epidermal growth factor receptor; KRAS: Kirsten rat sarcoma 2 viral
oncogene homolog; NMR: Nuclear magnetic resonance; FT: fourier-transform; IR:
Infrared; HRMS: High Resolution Mass Spectrometry; TLC: Thin layer
chromatography; PI: Propidium Iodide; ECL: Enhanced chemiluminescence; PARP:
Poly (ADP-ribose) polymerase; BAK: Bcl-2 homologous antagonist/killer protein;
BAX: BCL2 associated X protein; PUMA: p53 upregulated modulator of apoptosis.

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Introduction
Cancer is the second leading death cause in the world, and among all the cancer types,
colon cancer is one of the most lethal cancers which resulted in estimated over 50
thousand deaths in 2018 in the US according to the American Cancer Society report.
Even though significant advances have been made in screening techniques and
therapeutic agents for colon cancer, the treatment outcome remains poor for late
stages or metastatic patients. [1, 2] Moreover, genetic mutations aggravate the
challenge for colon cancer clinical treatment. [3-7] For example, approximately
30%-45% of colorectal cancer patients were reported to carry Kirsten rat sarcoma 2
viral oncogene homolog (KRAS) mutant (Mut) oncogenes which would lead to
failure of the epidermal growth factor receptor (EGFR) targeted therapies. [3, 8]
Therefore, identification of biomarkers and development of novel agents for colon
cancer suppression are in need.
Coumarins, natural secondary metabolites containing phenolic substances originally
discovered in various plant families, are consisted of a fused benzene with an
alpha-pyrone ring. [9] These compounds were firstly found in tonka bean and then
found exist in different kinds of plants such as vanilla and woodruff, in human dietary
components such as citrus fruits, green tea, and cinnamon products, or in essential oils
such as cinnamon bark and lavender oils, etc. [9-11] Literatures suggested that natural
coumarins and synthetic derivatives exhibit a variety of biological activities with
pharmacologic potential, include anti-bacterial, anti-inflammatory, anti-oxidant, and
anti-tumor capacities, etc. [12-17] Various coumarin derivatives with pharmacological
activities have been successfully applied in the clinical sites. [17-20]
Coumarin-based pharmacophoric substituents were reported under development for
anti-cancer agents by medicinal chemists.[15, 20-22] Among the numerous bioactive
coumarin derivatives, it was demonstrated that coumarin, 7-hydroxycoumarin, and
6-nitro-7-hydroxycoumarin exhibit potent anti-proliferation capacity towards different
cancer cells.[17, 23, 24] Also, 6-methoxy-7-hydroxycoumarin could induce apoptosis
pathway to suppress leukemic cell growth.[25] In this study, five coumarin derivatives
were synthesized, and the anti-cancer potential of these compounds was examined in
human cell lines, include colon cancer cells, breast cancer cells, lung cancer cells, and
non-cancerous cells, and then, molecular regulations and tumor inhibition
mechanisms of the coumarin compounds were investigated.
Material and Method

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Chemical synthesis of coumarin compounds: general experimental conditions
Melting points were determined in a capillary tube using a MEL-TEMP II melting
point apparatus by Laboratory Devices. Nuclear magnetic resonance (NMR)
spectroscopy was recorded on Bruker DMX-500 fourier-transform (FT)-NMR
spectrometers; chemical shifts were recorded in parts per million downfield from
Me4Si. Infrared (IR) spectroscopy was determined with a Perkin-Elmer 1760-X
FT-IR spectrometer. Mass spectra was recorded on Jeol JMS-D300 and FINNIGAN
TSQ-46C mass spectrometers; High Resolution Mass Spectrometry (HRMS) was
obtained with a Jeol JMSHX110 spectrometer. Thin layer chromatography (TLC) was
performed on Merck (Art. 5715) silica gel plates and visualized under UV light (254
nm), upon treatment with iodine vapor, or upon heating after treatment with 5%
phosphomolybdic acid in ethanol. Flash chromatography was performed with Merck
(Art. 9385) 40–63 lm silica gel 60. Reverse-phase high performance liquid
chromatography (RP-HPLC) analysis of the compounds was conducted as the
following method: Column: ACE 5 C18 4.6 x 250 mm, 5 µm; wavelength: 254 nm;
flow: 1.0 mL/min; mobile phase: CH3OH: 1% TFA.
5,7-Dihydroxy-4-methyl-chromen-2-one (9)
Concentrated H2SO4 (0.2 mL) was added to a mixture of phloroglucinol (6, 1.0 g, 7.9
mmol) and ethyl acetoacetate (7, 1.9 mL, 15.3 mmol) in glacial HOAc (6 mL) and
stirred for 2 h at room temperature. The precipitate was filtered, washed with water,
and crystallized with MeOH to give the title product. 1H NMR (500 MHz, DMSO-d6)
δ
2.48 (s, 3H), 5.83 (s, 1H), 6.15 (d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 10.28 (s,
1H), 10.51 (s, 1H).
5,7-Dimethoxy-4-methyl-chromen-2-one (10)
Methyl iodide (0.54 mL, 8.7 mmol) was added to a mixture of 9 (0.5 g, 2.9 mmol) and
potassium carbonate (0.8 g, 5.8 mmol) in anhydrous acetone (20 mL). Then reflux the
mixture for 2 h. The mixture was cooled at room temperature and added dist. water,
and filtration, washed with water. The crude product was crystallized with MeOH to
give the product 10. 1H NMR (500 MHz, CD3OD)
δ 2.56 (d, J = 1.0 Hz, 3H), 3.88 (s,
3H), 3.90 (s, 3H), 5.96 (d, J = 1.0 Hz, 1H), 6.26 (d, J = 2.0 Hz, 1H), 6.51 (d, J = 2.0
Hz, 1H).
5,7-Dihydroxy-4-trifluormethyl-chromen-2-one (1)
Concentrated H2SO4 (0.5 mL) was added to a mixture of phloroglucinol (6, 5.0 g,
39.6 mmol) and ethyl trifluoroacetoacetate (8, 8.6 mL, 47.5 mmol) in glacial HOAc

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(20 mL) and heated at 100oC for 2 h. The reaction mixture was cooled at room
temperature and diluted with water. The precipitate was filtered, washed with water,
and crystallized with MeOH to give the title product. 95.0% purity (mobile phase:
1
CH3OH: 1% TFA = 1:1, injection: 20 µL, tR: 15.0 min). H NMR (500 MHz,
DMSO-d6)
δ 6.28 (d, J = 2.5 Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H), 6.53 (s, 1H), 10.66 (s,
1H), 10.91 (s, 1H).
5,7-Dimethoxy-4-trifluoromethyl-chromen-2-one (2)
The title compound was obtained from compound 1 in a similar way as described
compound 10. 99.3% purity (mobile phase: CH3OH: 1% TFA = 7:3, injection: 20 µL,
tR: 8.9 min). 1H NMR (500 MHz, DMSO-d6)
= 2.5 Hz, 1H), 6.72 (d, J = 2.5 Hz, 1H). 13C NMR (125 MHz,
DMSO-d6) 56.20, 56.70, 94.55, 96.37, 98.37, 113.10 (q, J = 32.3 Hz), 121.75 (q, J
δ 3.89 (s, 3H), 3.89 (s, 3H), 6.62 (d, J
δ
= 1090.7 Hz), 138.77 (q, J = 133.9 Hz), 156.73, 156.91, 158.69, 163.87.
5,7-Dimethoxy-4-methyl-3-nitro-chromen-2-one (3) and
5,7-Dimethoxy-4-methyl-6-nitro-chromen-2-one (4)
To a mixture of 5,7-Dimethoxy-4-methyl-chromen-2-one (10, 0.1 g, 0.45 mmol) and
Cr(NO3)2·9H2O (0.22 g, 0.5 mmol) in acetic anhydride (5 mL) was stirred at room
temperature for 2 h. After the completion of the reaction, as monitored on TLC, the
reaction mixture was filtered and the filtrate was diluted with water. The contents
were extracted with ethyl acetate and dried over anhydrous MgSO4 and filtration. The
solvent was removed by distillation under reduced pressure and the resulting crude
product was subjected to column chromatography (EtOAc/n-hexane = 1/10) to give 3
and 4.
3: 98.1% purity (mobile phase: CH3OH: 1% TFA = 7:3, injection: 20 µL, tR: 6.7 min).
1H NMR (500 MHz, DMSO-d6)
δ
2.50 (s, 3H), 3.89 (s, 3H), 3.91 (s, 3H), 6.63 (d, J =
2.0 Hz, 1H), 6.74 (d, J = 2.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6)
δ
18.16, 56.33,
56.74, 94.19, 96.84, 101.60, 134.70, 147.30, 153.17, 155.06, 160.70, 164.76; MS (EI,
+
70 eV) m/z 266 (M+H+), 157 (base peak); HRMS (EI) calculated for C12H12NO6 :
266.0659, found: 266.0660.
4: 96.4% purity (mobile phase: CH3OH: 1% TFA = 6:4, injection: 10 µL, tR: 6.3 min).
1H NMR (500 MHz, CDCl3/DMSO-d6)
δ
2.51 (s, 3H), 4.00 (s, 3H), 4.03 (s, 3H), 6.11
23.66, 56.89, 57.28,
13
(s, 1H), 6.75 (s, 1H); C NMR (125 MHz, CDCl3/DMSO-d6)
δ
92.56, 103.33, 111.44, 123.54, 147.03, 153.80, 154.02, 157.29, 160.08; MS (EI, 70
+
eV) m/z 266 (M+H+), 157 (base peak); HRMS (EI) calculated for C12H12NO6 :
266.0659, found: 266.0660.

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6-amino-5,7-Dimethoxy-4-methyl-chromen-2-one (5)
To a mixture of 4 (0.12 g, 0.45 mmol), Fe (0.14 g, 2.5 mmol), and NH4Cl (0.13 g, 2.5
mmol) in 75% EtOH (10 mL) was refluxed for 1 h. The reaction mixture was filtrated
by celite-aided, then evaporated to remove EtOH. The residue was diluted with water,
basified with 10% NaOH(aq) to pH 10-11, and extracted with ethyl acetate. The
organic layer was dried (anhydrous MgSO4) and filtered. The solvent was removed by
distillation under reduced pressure and the resulting crude product was subjected to
column chromatography (MeOH/CH2Cl2 = 1/10) to give 5. 99.3% purity (mobile
1
phase: CH3OH: 1% TFA = 1:1, injection: 20 µL, tR: 4.3 min). H NMR (500 MHz,
13
DMSO-d6)
δ
2.51 (s, 3H), 3.84 (s, 3H), 3.92 (s, 3H), 5.98 (s, 1H), 6.64 (s, 1H); C
NMR (125 MHz, DMSO-d6)
δ
23.69, 56.11, 56.60, 93.36, 103.89, 110.40, 118.27,
141.42, 149.12, 149.57, 154.53, 159.55; MS (EI, 70 eV) m/z 236 (M+H+), 157 (base
+
peak); HRMS (EI) calculated for C12H14NO4 : 236.0917, found: 236.0917.
Cell culture
Human cell lines were from ATCC. Colon cancer cell lines, DLD-1 and HCT116 were
sustained in RPMI medium (Gibco). Colon cancer HT29 cells, breast cancer cell lines,
MDA231 and MCF7, lung cancer cell lines, A549 and H1299, and IMR90 lung cells
were cultured in DMEM medium (Gibco). Cell culture medium was supplemented
with 5-10% Fetal bovine serum (Gibco) as well as 1% antibiotic (Gibco) and
incubated at 37℃ with 5% CO2.
Colony formation assay
Cells were seeded in the 6 well plates with 100 cells in each well, and the next day,
different treatments were applied. 14 days later, colonies were examined by fixing and
staining the cells with 0.5% crystal violet in acetic acid/ methanol solution. After the
staining, plates were rinse with water and dried for colony counting. The experiment
was performed in triplicate in each group. [26]
Wound healing assay
Cells were seed in 6 or 12 well plates for next day over 90% cell confluence. Then,
treatments were done, scratches (wounds) were made, and cell movement capacity
was monitored under the microscope at indicated time points. Migration rate of the
cells was examined and determined as (distance at 0hr – distance at each time point) /
distance at 0hr.
Live and dead cell assay
Calcein-AM (Cayman) and Propidium Iodide (PI, Omics Bio) were dissolved in

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DMSO and diluted in PBS. Cells were seeded in the 96 well plates, and then the
treatments were applied on the next day. 48hr later, the medium in each well was
removed, and 6µM Calcein-AM and 6µM PI were added as 1:1 ratio into each well
for cell staining. At the end, cells were monitored under the immunofluorescence
microscope at 490nm.
Western blot assay
DLD-1 cells were treated with coumarin compound #4 and harvested at indicated time
points. Samples were analyzed by western blot assay as described.[26, 27] In brief,
samples were lysed, and proteins were extracted, separated by SDS polyacrylamide
gel electrophoresis, and transferred to PVDF membrane. Primary antibodies
(GeneTex) probing for specific proteins were used as indicated. Signals were detected
by Enhanced chemiluminescence (ECL) and analysis. Expression of proteins relative
to control was analyzed by image J software.
MTT cell proliferation assay
Cells were seeded in 96 well plates, and treatments were applied on the next day. Cell
viability was then analyzed at indicated time points by MTT((3-(4,
5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) assay (Goldbio) as
described.[28] EC50 was analyzed and determined by SigmaPlot software. The
experiment was performed in at least triplicate in each group.
Statistical analysis
Representative data were shown, and all the experiments were repeated in at least
triplicate in each condition. Data were analyzed by one-way ANOVA or student t-test
and showed as mean ± SD. P value <0.05 was taken as significant data and marked as
“*.”
Results
Synthetic scheme of coumarin derivatives
The coumarins 1-5 were synthesized as in Figure 1. Phloroglucinol (6) treated with
ethyl acetoacetate/trifluoroacetoacetate (7/8) under glacial acetic acid and catalyzed
by
conc.
H2SO4
to
give
5,7-dihydroxy-4-methylcoumarin
9
and
5,7-dihydroxy-4-trifluoromethylcoumarin 1. Methylation of 9 and 1 yielded
5,7-dimethoxycoumarin 10 and 2, and then nitration of 10 obtained
6-nitro-5,7-dimethoxy-4-methylcoumarin 4 and 3-nitrocoumarin 3. Reduction of
nitro-coumarin 4 yielded amino-coumarin 5 (Figure 1). The purity of compounds was
then determined by HPLC. The purities of all five compounds were higher than 95%,

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and the data were shown in Supplementary Figure 1. These five coumarin compounds
were named by order (Figure 1a) as #1 to #5 in the following experiments.
The coumarin derivatives showed specific cytotoxicity towards colon cancer cells
As mentioned, anti-cancer potential of synthetic coumarin derivatives has been
suggested in cancer cells before. Therefore, we firstly examined our five compounds
in different cancer cell types, to investigate their cancer inhibition capacity in various
cancers. Colon, breast, and lung cancer cell lines were treated with 1, 10, or 100µM of
these compounds, and then cell viability was examined 72 hours later by MTT assay.
The results showed that among these five compounds, compound #4 exhibited the
strongest and specific anti-cancer capacity towards colon cancer cells, in both DLD-1
and HT29 cells (Figure 2a and 2b), rather than in other cancer types (Figure 2c-2f) or
in non-cancerous IMR90 cells (Figure 2g). Even though not as powerful as #4,
compound #3 also exhibited certain anti-cancer effectiveness in breast cancer cells
(Figure 2c and 2d). Also, generally speaking, compound #3, #4, and #5 exhibited
better dose dependent cytotoxicity towards colon cancer cells then in breast or lung
cancer cells (Figure 2a-2f). Our results suggest that compound #4 could inhibit colon
cancer cells with high specificity (Figure 2).
Coumarin derivatives can serve as proliferation inhibitors to KRAS mutant
colon cancer cells
Among the above two different colon cancer cells, DLD-1 cells carry Mut KRAS
gene while as HT29 cells carry wild type (WT) KRAS gene. In order to examine and
confirm whether our coumarin compound #4 could suppress Mut KRAS colon cancer,
we applied HCT116, another Mut KRAS colon cancer cell line, in the study. Next, we
had a closer cancer inhibition investigation towards compounds #3, #4, and #5 in
DLD-1 and HCT116 cells. Cells were treated with different concentrations of
compounds, from 0, 5, 10, 20, to 40µM, and then cell viability was examined at 48hr
and 72hr time points by MTT assay. The results showed that among these three
compounds, compound #4 exhibited best cancer inhibition capacity in both DLD-1
(Figure 3a-3c) and HCT116 cells (Figure 3d-3f), and therefore compound #4 was
further investigated in the following experiments. Cell morphology was monitored
and recorded (Figure 3c and 3f).
Compound #4 suppressed short term and long term proliferation of colon cancer
cells
To better understand how compound #4 inhibits colon cancer cells, we examined it
anti-cancer effect in short term MTT proliferation assay, identified the EC50 (or IC50)

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concentration of compound #4, and then in colony formation assay for investigation
of their long term effect. Cells were treated with 0, 2.5, 5, 7.5, or 10µM of compound
#4, and then examined by MTT assay at 48hr time point. The results showed that
compound #4 exhibited dose dependent cancer inhibition effect in both DLD-1 and
HCT116 cells (Figure 4a and 4c). EC50 of compound #4 was also examined for these
two cell lines as about 6.5µM for DLD-1 cells and nearly 7.7µM for HCT116 cells
(Figure 4b and 4d). Next, colony formation assay was carried out under the treatments
with 0, 5, 10, and 20µM of compound #4 (Figure 4e). Numbers of colonies were
organized and normalized to untreated control group in Figure 4f as a bar chart. The
results indicate that compound #4 could suppress short term and long term colon
cancer proliferation. Also, DLD-1 cells seemed to be more sensitive to the coumarin
compound #4 compared to HCT116 cells (Figure 3 and 4), and therefore DLD-1 cells
were applied for the following assays.
Compound #4 inhibited the migration of colon cancer cells
Metastasis is one of the major concerns for clinical colorectal cancer treatment, and
high migration capacity of colon cancer cells could at the end result in lung or liver
metastasis [29]. Therefore, we next investigated whether our coumarin compound can
impact on the migration ability of colon cancer cells. Wound healing assay was
performed in cells to examine the migration inhibition potential of compound #4 at 0,
5, 10, or 50µM concentrations. The results showed that compared to untreated control
group, compound #4 could significantly slow down the migration of DLD-1 cells at
10µM and 50µM (Figure 5a). The wound recovery rates or migration rates of different
groups were organized as a bar chart (Figure 5b). When incubated with 50 µM
compound #4, DLD-1 cells did not really move during the whole experimental period
of time. The data suggest that compound #4 exhibits the migration inhibition capacity
of colon cancer cells.
Compound #4 induced apoptotic cell death of colon cancer cells
Our data suggested that compound #4 exhibits specific cytotoxicity effect towards
colon cancer cells (Figure 2). In order to better understand the molecular mechanism
of cell death caused by this coumarin compound, we continued to investigate whether
compound #4 induces colon cancer death through apoptosis pathway. First, live and
dead assay was carried out by applying Calcein-AM and PI staining as described in
materials and methods. After 48hr treatments with different concentrations of
compound #4, cells were stained, and then live cells with green fluorescence emission,
or dead cells with red emission, were monitored under the fluorescence microscope
(Figure 6a). The intensity of red fluorescence increased along with the increase of

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compound #4, confirming that compound #4 kills colon cancer cells with a dose
dependent fashion (Figure 6a). According to the previous study, coumarin derivatives
could exhibit anti-tumor effect and induce apoptosis in cancer cells [30]. Therefore,
we continued to examine whether compound #4 induce cytotoxicity of colon cancer
cells through upregulating the apoptosis pathway. Cells were treated with compound
#4 at 0, 5, or 10 µM concentrations, and then harvested at 24hr, 48hr, or 72hr time
points for western blot analysis. Regulation of apoptotic markers include Poly
(ADP-ribose) polymerase (PARP), Bcl-2 homologous antagonist/killer protein (BAK),
BCL2 associated X protein (BAX), and p53 upregulated modulator of apoptosis
(PUMA), were examined. The results showed that treatment of compound #4 resulted
in PAPR cleavage and induction of BAK and PUMA proteins (Figure 6b). The
expression of apoptotic proteins relative to controls was analyzed as a bar chart
(Figure 6c). The induction of BAK can be observed in different time points; the pick
expression of cleaved PARP fell in the 10µM 48hr time point; the pick expression of
PUMA fell in the 10µM 24hr time point; slight elevation of BAX was observed after
the treatments (Figure 6b). The data suggest that compound #4 carried out its
anti-tumor capacity through activation of apoptosis signaling pathway.
Summary and Discussion
Colon cancer was targeted by other coumarin derivatives previously. Osthol, one of
the coumarin derivatives with methoxy on the C7, was reported as a strong anti-tumor
compound. Among several different types of tumors, Osthol seemed to exhibit tissue
specific effect towards colon cancer cells (HCT116 and SW480 cells)[31], of which
the dose of Osthol applied was much lower than in other cancer cells, such as prostate
and breast cancer cells. Compatibly in our study, colon cancer cells were generally
more sensitive to our coumarin compounds, which have the methoxy instead of
hydroxyl group, compared to other cancer types (Figure 2).
According to our data, among the five coumarin derivatives examined in this study,
compounds #3 and #4 contain nitro group and methoxy group instead of
trifluoromethyl group (in compound #1 and #2), another frequently used backbone for
anti-tumor effect in coumarin derivatives studies [32, 33], were more effective in
colon cancer suppression (Figure 1 and 2). It suggested that nitro- substitution,
compared to trifluoromethyl- substitution, could lead to a better cancer inhibition
performance. Moreover, the anti-cancer potential of nitro- and/or hydroxyl-coumarin
derivatives,
such
as
6-nitro-7-hydroxycoumarin,
7,8-dihydroxycoumarin,
3,6,8-nitro-7-hydroxycoumarin, and 8-nitro-7-hydroxycoumarin, were previously
investigated in kidney, skin, and lung cancer cells, respectively. [34-36] The

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comparison of 3- or 6-substitution position of NO2 on the compound (compound #3 or
#4) also suggested that 6-NO2 substitution coumarin could result in higher
cytotoxicity efficiency, and that 6-amino- substitution does not seem to be a promising
anti-cancer option (Figure 1 and 3) for colon cancer treatment. Also, between
compound #3, #4, and #5, the structure of compounds #5 is similar to the other two
but contain NH2 rather than NO2 functional group (Figure 1). The cancer cell
inhibition data suggested that NO2 functional group is more powerful than NH2 on a
cumoarin derivative for producing anti-cancer capacity (Figure 3).
Coumarin derivatives were reported to induce anti-proliferative effect and cell
apoptosis toward cancer cells previously, and one of the regulated apoptotic pathways
was mediated through mitochondria. [5] Compatibly, in this study, we discovered that
our coumarin compound #4 induced cell death in a dose dependent manner (Figure
6a). The data also suggest that compound #4 induced cytotoxicity of colon cancer
cells through facilitating the intrinsic and extrinsic apoptosis pathways with activation
or elevation of several apoptotic markers, include PARP, BAK, BAX, and PUMA
(Figure 6b).
As mentioned, one of the famous mutations in tumors, KRAS mutation, could lead to
inefficiency of colon cancer treatment in patients under EGFR monoclonal antibody
targeted therapies.[8] Coumarin derivatives were reported to suppress lung cancer
cells harboring Mut KRAS gene before.[37] Here, in this study, we applied three
colon cancer cells lines for demonstrating the cytotoxicity effect of coumarin
compound #4, and among them, HT29 cells contain Wt KRAS gene, while as DLD-1
and HCT116 cells harbor mutated KRAS genes, suggested that colon cancers with
either Wt or Mut KRAS genes can potentially be treated by our coumarin compound
#4. Moreover, in terms of the tissue specific effect of coumarin compound #4 and
whether coumarin compound #4 could suppress other cancer types with mutated
KRAS gene, our data also provided some suggestions. In Figure 2, we applied cancer
cell lines harboring Wt or Mut KRAS. Colon cancer cells DLD1 (Mut), and HT29
(Wt); breast cancer cells MDA231 (Mut) and MCF7 (Wt); lung cancer cells A549
(Mut) and H1299 (Wt). Our data suggest that coumarin derivate #4 targeted with high
specificity towards colon cancer carrying either Wt or Mut KRAS gene, rather than
towards other cancer cells.
Finally, our data showed efficient anti-cancer effect of compound #4 towards these
cells with low EC50 (Figure 4), indicating that compound #4 is a promising lead drug
for colon cancer inhibition. Further in vivo investigation in the future would be helpful

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for confirming the anti-tumor drug development potential of compound #4.
Acknowledgments
This project was partly supported by the Ministry of Science and Technology (MOST
107-2320-B-038-054, 107-2813-C-038-108-B), Taiwan, and WanFang Hospital,
Chi-Mei Medical Center, and Hualien Tzu-Chi Hospital Joint Cancer Center
Grant-Focus on Colon Cancer Research (MOHW108-TDU-B-212-124020, supported
by Health and welfare surcharge of tobacco products), Taiwan. We thank Shwu-Huey
Wang for help with the NMR spectra and data calculation, and we also appreciate the
supports from the Core Facility Center at Taipei Medical University, Taiwan.
Conflict of interest
None.
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Figure Legends
Figure 1. Coumarin derivatives and synthetic scheme. Coumarin derivatives 1-5 (a)
and synthetic scheme of coumarins 3-5 (b) are shown here. Reagents and conditions: a:
glacial HOAc, H2SO4, RT, 90%; b: K2CO3, CH3I, anhydrous Acetone, reflux, 87.5%; c:
Cr(NO3)3·9H2O, anhydrous acetic anhydride, RT, 59% (3: 26%, 4: 33%); d: Fe,
NH4Cl, 75% EtOH, reflux, 75%.
Figure 2. Examination of the cytotoxicity of coumarin derivatives compounds
towards different cancers. Different cancer cells were applied treated with 0, 1,
10,100 µM of coumarin derivatives compounds for 72hr, and then cell viability was
examined by MTT assay. Colon cancer cells (a) DLD-1 and (b) HT29, breast cancer
cells, (c) MDA231 and (d) MCF, lung cancer cells, (e) A549 and (f) H1299, or
non-cancerous human cells IMR90, were treated with compound #1-#5 in this
experiment. n=3.
Figure 3. Coumarin derivatives served as proliferation inhibitor to KRAS
mutant colon cancer cells. Coumarin compounds #3-5 were applied for treating
DLD-1 cells (a)-(c) or HCT116 cells (d)-(f). n=2 for (a)(d), and n=3 for (b)(e). Cell
viability was examined at indicated time points, and cells were also monitored under
the bright field microscope at 72hr time point (c) and (f).
Figure 4. Compound #4 suppressed short term and long term proliferation of
colon cancer cells. DLD-1 cells (a)-(b) and HCT116 cells (c)-(d) were treated with
indicated concentrations of compound #4, cell viability was analyzed at 48hr time
point by MTT assay, and EC50 was determined by SigmaPlot software (b) and (d).
n=3 for (a)-(d). (e) DLD-1 cells were applied in colony formation assay under the
treatment of compound #4 with indicated concentrations. Colony numbers were
organized in (f) as a bar chart. n=2.
Figure 5. Compound #4 inhibited the migration of colon cancer cells. Wound
healing assay was perform in DLD-1 cells under compound #4 treatment at indicated
concentrations. After the scratches were made, cell migration was monitored at 0hr,
4hr, 8hr, 24hr time points under the microscope (a) and the migration rate (wound
recovery) was organized in (b) as a bar chart. n=2.

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Figure 6. Compound #4 induced apoptotic cell death of colon cancer cells. (a)
DLD-1 cells were treated with different concentrations of compound #4, and cells
were analyzed by live and dead assay and then examined by fluorescence microscope.
(b) DLD-1 cells treated with different concentrations of compound #4 were harvested
at indicated time points. Specific protein expression of these samples was analyzed by
western blot. GAPDH and vinculin were used as loading control. (c) Expression of
proteins relative to control was analyzed, and cleaved PARP was analyzed here. n=2.
Supplementary Figure 1. Determined the purity of compounds 1-5 by HPLC. The
conditions applied were shown as below: Hitach L2420; UV-Vis detector: 254 nm;
Column: ACE 5 C18, 4.6 x 250 mm, 5 µm; Concentration: 1 mg/mL; Flow rate: 1
mL/min; Mobile phase: CH3OH: 1% TFA. (a) Compound 1. (b) Compound 2. (c)
Compound 3. (d) Compound 4. (e) Compound 5.

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Highlights
ò
Coumarin derivatives were synthesized and examined in different human cancer
cells.
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ò
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A novel compound exhibited cytotoxicity specifically towards colon cancer cells.
It can inhibit short-term and long-term cancer cell proliferation.
It can suppress migration capacity of colon cancer cells.
It can facilitate cancer cell death through activation of apoptosis pathway.