Biological evaluation of non-basic chalcone CYB-2 as a dual ABCG2/ABCB1 inhibitor

Article abstract of DOI:10.1016/j.bcp.2020.113848

The enhancement of drug efflux caused by ATP-binding cassette (ABC) transporters (including ABCG2 and ABCB1) overexpression is an important factor for multidrug resistance (MDR) in cancers. After testing the reversal activities of 19 chalcone and bis-chal

Full text of DOI:10.1016/j.bcp.2020.113848

Journal Pre-proofs
Biological evaluation of non-basic chalcone CYB-2 as a dual ABCG2/ABCB1
inhibitor
Chao-Yun Cai, Wei Zhang, Jing-Quan Wang, Zi-Ning Lei, Yun-Kai Zhang, Yi-
Jun Wang, Pranav Gupta, Cai-Ping Tan, Bo Wang, Zhe-Sheng Chen
PII:
S0006-2952(20)30069-1
https://doi.org/10.1016/j.bcp.2020.113848
BCP 113848
DOI:
Reference:
Biochemical Pharmacology
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Accepted Date:
7 December 2019
5 February 2020
Please cite this article as: C-Y. Cai, W. Zhang, J-Q. Wang, Z-N. Lei, Y-K. Zhang, Y-J. Wang, P. Gupta, C-P. Tan,
B. Wang, Z-S. Chen, Biological evaluation of non-basic chalcone CYB-2 as a dual ABCG2/ABCB1 inhibitor,
Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.2020.113848
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Biological evaluation of non-basic chalcone CYB-2 as a dual
ABCG2/ABCB1 inhibitor
Chao-Yun Caia, b, Wei Zhang a, c, Jing-Quan Wang a, Zi-Ning Lei a, Yun-Kai Zhang a, Yi-Jun
Wang a, Pranav Gupta a, Cai-Ping Tanb, *, Bo Wang b, *, Zhe-Sheng Chen a, *
a Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s
University, 8000 Utopia Parkway, Queens, New York 11439, United States
b MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-
sen University, 135 Xingang West Road, Guangzhou, 510275, P. R. China
c Institute of Plastic Surgery, Weifang Medical University, Weifang, Shandong 261041, P. R.
China
*Corresponding author
Abstract
The enhancement of drug efflux caused by ATP-binding cassette (ABC) transporters
(including ABCG2 and ABCB1) overexpression is an important factor for multidrug
resistance (MDR) in cancers. After testing the reversal activities of 19 chalcone and bis-
chalcone derivatives on MDR cancer cell lines, we found that non-basic chalcone CYB-2
exhibited the most potent reversal activities against both ABCG2- and ABCB1-mediated
MDR. The mechanistic studies show that this compound can increase the accumulation
of anticancer drugs in both ABCG2- and ABCB1-overexpressing cancer cell lines,
resulting from the blocked efflux function of the MDR cancer cell lines. This inhibition is
due to the barred ABCG2 and ABCB1 ATPase activities rather than altering the
1

expression or localization of ABCG2 or ABCB1 transporters. The previous studies
showed that non-basic chalcones were ABCG2-specific inhibitors; however, we found
that non-basic chalcone CYB-2 can be developed as an ABCG2/ABCB1 dual inhibitor to
overcome MDR in cancers that co-express both ABCG2 and ABCB1. Moreover, non-
basic chalcone CYB-2 has synthetic tractability compared to other chalcone-based
derivatives.
Keywords
Multidrug resistance (MDR); Chalcone derivatives; ABCG2; ABCB1; ABC transporters
1. Introduction
The failure of chemotherapy is mainly caused by multidrug resistance (MDR), which
involves a variety of cellular pathways. There are many factors that contribute to MDR in
cancer, including the reduction of the intracellular accumulation and increase of drug
efflux [1], enhanced DNA damage repair [2], cell death inhibition, epithelial-
mesenchymal transition [3], drug target alteration [4], and the enhancement of drug
inactivation [5]. The increase of drug efflux caused by the overexpression of ATP-
binding cassette (ABC) transporters is an important contributor to MDR in cancer cells.
ABC transporters with seven subfamilies (ABCA~ABCG) are a superfamily of 48
transporters expressed in humans, of which ABCE and ABCF subfamilies have no
membrane transport functions [6]. ABC transporters consist of ABCG2 (breast cancer
resistance protein/BCRP; mitoxantrone (MX)-resistant gene/MXR), ABCB1 (P-
glycoprotein/P-gp). A large number of anticancer drugs are substrates of ABC
transporters; thus these drugs can be pumped out of cancer cells that express ABC
2

transporters, subsequently decreasing in efficacy. Some well-known substrates for the
ABC transporters are as follows: ABCG2 substrates (doxorubicin, mitoxantrone, SN-38,
topotecan et. al.) [7]; ABCB1 substrates (colchicine, actinomycin-D, doxorubicin,
paclitaxel, vinblastine, vincristine et. al.) [8].
It was also reported that chalcones with α,β-unsaturated carbonyl structure has
pharmacological significance exhibiting various biological activities [9,10], e.g.,
anticancer [11–13], antimicrobial [14,15], anti-inflammatory, and antioxidant [16]
properties. The heterocyclic ring system of chalcones can be used as excellent ligands in
metal complexes, with various biomedical activities [17]. Several chalcone-based
compounds have been approved and marketed [18,19], such as metochalcone (choleretic
and diuretic agent), sofalcone (antiulcer and mucoprotective) and hesperidin
methylchalcone (venous insufficiency treatment) [20], which reveals the varied utility of
chalcone scaffolds in drug development. Some compounds with a chalcone scaffold, such
as chalcone- and flavone-based compounds [21], quinazoline-chalcone compounds [22],
bifendate-chalcone hybrids [23], and indole-basic chalcone [24], were found to be
effective agents that reverse ABCG2- and/or ABCB1-mediated MDR. It was also
reported that bis-chalcone with two α, β-unsaturated carbonyl system can reverse
ABCG2-mediated MDR [25]. It was also reported that some non-basic chalcones can
selectively reverse ABCG2-mediated MDR [26,27]. In previous reports, the chalcone
compounds that inhibited both ABCG2 and ABCB1 were chalcone-based derivatives
rather than non-basic chalcones.
3

The previous reports showed that some cancers overexpress both ABCG2 and
ABCB1 [28]. The cancers co-expressing multiple transporters may cause more potent
resistance to anticancer drugs than cancers overexpressing a single transporter [29].
Herein, we tested some non-basic methoxy and hydroxy chalcones to discover new
chalcone derivatives that act as dual inhibitors of potent ABCG2 and ABCB1. We also
included bis-chalcones to reveal whether the extended π system is crucial in increasing
the reversal effects. We conducted the drug accumulation and efflux assay, Western
blotting, immunofluorescence, ATPase assay and docking analysis to reveal the
mechanism of chalcone derivatives reversing MDR in cancers.
2. Materials and methods
2.1. Chemistry
The chemicals and reagents for synthesis were purchased from Alfa Aesar (Haverhill,
MA.) or Tokyo Chemical Industry (Tokyo, Japan). The synthesis of the chalcone
derivatives except CYB-2, CYB-7, CYB-15 had been reported in our previous study
[30]. CYB-2, CYB-7, and CYB-15 were synthesized through the same method as the
other chalcone derivatives. The methoxy chalcones CYB-2 and CYB-7 were synthesized
by the following procedures: benzaldehydes and acetyl benzenes were dissolved in
methanol and aqueous KOH (50%) was added. After 3h reaction at 70℃, the solution
was poured into water to form precipitates. Methanol was evaporated under reduced
pressure. After extraction with CH2Cl2 and the evaporation, the raw product was purified
by flash chromatography. CYB-15 was synthesized through the demethylation of CYB-
13 with boron tribromide (BBr3). The reaction underwent 24 h at room temperature, and
4

then the mixture was poured into ice water. The precipitate was purified by flash
chromatography.
2.2. Cell culture
In this study, we used the human colorectal cancer cell line S1, the human lung cancer
cell line NCI-H460 and S1-M1-80 (MX-selected ABCG2-overexpressing cell line of S1)
[31], NCI-H460/MX20 (MX-selected ABCG2-overexpressing cell line of NCI-H460)
[32]. HEK293/pcDNA3.1 is a human embryonic kidney 293 cell line with a pcDNA3.1
vector, while HEK/ABCG2-482-R2, HEK/ABCB1 were established with a pcDNA3.1
vector containing ABCG2-482-R2 and ABCB1. The transfected cell lines were
maintained in medium with G418 (2 mg/mL) [33]. The KB-C2 cell line was selected by
colchicine (Alfa Aesar, Haverhill, MA.) (2 μg/mL) with human cervical carcinoma cell
line KB-3-1 [34]. The cell lines were cultured with Dulbecco's Modified Eagle Medium
(Corning Inc., Corning, NY) under the condition of 37°C and 5% CO2.
2.3. MTT assay
Cytotoxicity tests, inhibition and reversal effect assays were performed using the 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Biosynth International,
Inc., Itasca, IL) assay. Cells were seeded 24h prior to the addition of the drugs. For the
MDR reversal activities study, chalcones or positive controls were pre-incubated 2 h
prior to the addition of the chemotherapeutic drugs. After 72 h, the MTT reagent (4
mg/mL) was added for 4 h incubation. Subsequently, the supernatant was removed and
100 μL of dimethyl sulfoxide (DMSO) (MilliporeSigma Company, Burlington, MA) was
5

added to dissolve the purple crystals. The absorbance was measured with a UV/Vis
microplate spectrophotometer (Fisher Scientific International, Inc., Hampton, NH).
2.4 Tritium-labeled drugs accumulation and efflux assay
The drug accumulation assay was conducted as the previous report [35]. The reversal
agents were pre-incubated for 2 h, then the [3H]-MX (Moravek Biochemicals Inc., Brea,
CA) or [3H]-paclitaxel-containing (Moravek Biochemicals Inc., Brea, CA) medium was
added, in the presence or absence of reversal agents. After 2 h of incubation, the cell
lysates were added into scintillation fluid (5 mL). For the efflux assay, the procedures of
drug accumulation study are performed. Subsequently, the tritium-labeled drug and
reversal agents containing medium was removed and the cells were incubated with
medium containing only reversal agents. And then the cells were lysed at various time
points (0, 30, 60, and 120 min) and the cell lysates were added into 5 mL of scintillation
fluid. The radioactivity of the samples was measured with the Packard TRI-CARB1
190`A liquid scintillation analyzer.
2.5. Protein immunoblotting
The proteins were extracted from the cell lysates as described in previous reports [36].
Then the proteins were loaded into sodium dodecyl sulfate polyacrylamide gel and
resolved by electrophoresis, and then the resolved proteins were transferred onto
polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and then
incubated with the primary monoclonal antibodies against β-actin (Thermo Fisher
Scientific, Inc., Waltham, MA), GAPDH (Cell Signaling Technology, Inc., Danvers,
MA), ABCG2 (BXP 21) (Gene Tex Inc., Irvine, CA) or ABCB1 (F4) (Sigma-Aldrich,
6

Inc., St. Louis, MO) overnight at 4°C, followed by 2h incubation at room temperature
with anti-mouse IgG, horseradish peroxide (HRP)-linked antibody (Cell Signaling
Technology, Inc., Danvers, MA). The chemiluminescent signal was detected using ECL
detection reagents (MilliporeSigma Company, Burlington, MA) and captured on the film.
2.6. Immunofluorescence analysis
As described in the previous reports [37], the NCI-H460/MX20 and KB-C2 cells were
incubated with or without reversal agents. Thereafter, cells were fixed with 4%
paraformaldehyde for 15 min, followed by permeabilization with 1% triton X-100 for 10
min. The cells were blocked with 6% BSA for 1 h at 37 °C, and then were incubated with
monoclonal anti-ABCG2 protein (BXP 21) (Gene Tex Inc., Irvine, CA) or monoclonal
anti-ABCB1 antibody (F4) (Sigma-Aldrich Inc., St. Louis, MO) overnight at 4°C,
followed by 2 h incubation with Alexa flour 488 goat anti-mouse IgG (Abcam plc.,
Cambridge, United Kingdom) at 37°C. After that, the cells were stained with 4’,6-
diamidino-2-phenylindole (DAPI) or propidium iodide (PI). The images were captured
with a Nikon TE-2000S fluorescence microscope (Nikon Instruments Inc., Melville, NY).
2.7. ABCG2/ABCB1 ATPase assay
The ATPase assay was conducted with a PREDEASY ATPase assay kit (Sigma-Aldrich
Inc., St. Louis, MO) [38].
2.8. Molecular modeling
Molecular docking was performed in Maestro v11.1 (Schrꢀdinger, LLC, New York, NY,
2017) software [39]. The protein preparation of the wild-type human ABCG2 (PDB:
7

6FFC) [40] was performed and the grid was generated. The protein preparation and grid
generation for ABCB1 (PDB: 6FN1) [41]were performed with the same protocol. The
ligand was prepared and Glide docking was conducted and the best-scored conformation
of the ligand and protein were obtained. After that, induced-fit docking (IFD) was
performed with default parameters.
2.9. Statistical analysis
All experiments were conducted at least three times. The significance was analyzed by
two-way ANOVA, and P value <0.05 represents significant difference.
3. Results and discussion
3.1 Chemistry
The structural characterization of CYB-2, CYB-7, and CYB-15 are shown as
follows. CYB-15 is a new compound which has not been reported in the previous reports.
The methoxy chalcones can be synthesized easily via one-step reactions compared to the
other chalcone-based derivatives, which is an advantage for methoxy chalcones to be
developed as ABCG2/ABCB1 transporters inhibitors.
(E)-3-(2, 4-dimethoxyphenyl)-1-(3, 4-dimethoxyphenyl) prop-2-en-1-one (CYB-2)
Scheme 1. The structure of CYB-2
Yield 70.6 %, 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 15.7 Hz, 1H), 7.71 – 7.61 (m,
2H), 7.60 – 7.53 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.53 (dd, J = 8.6, 2.3 Hz, 1H), 6.47 (d,
J = 2.3 Hz, 1H), 3.96 (d, J = 7.2 Hz, 6H), 3.90 (s, 3H), 3.85 (s, 3H), 13C NMR (126 MHz,
CDCl3) δ 189.3, 162.9, 160.3, 152.9, 149.1, 139.7, 131.9, 130.8, 122.8, 120.1, 117.3,
110.9, 110.0, 105.4, 98.4, 56.0, 56.0, 55.5, 55.5
8

(2E, 2'E)-3, 3'-(1, 3-phenylene) bis (1-(3, 4-dimethoxyphenyl) prop-2-en-1-one) (CYB-7)
Scheme 2. The structure of CYB-7
Yield 86.0 %, mp 139~140℃. 1H NMR (400 MHz, DMSO) δ 8.45 (s, 1H), 8.09 (d, J =
15.6 Hz, 2H), 7.98 (d, J = 8.0 Hz, 4H), 7.79 (d, J = 15.6 Hz, 2H), 7.64 (s, 2H), 7.56 (t, J =
8.0 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 3.89 (d, J = 5.2 Hz, 12H).13C NMR (101 MHz,
CDCl3) δ 188.3, 153.5, 149.4, 143.0, 135.9, 131.2, 129.8, 129.5, 128.3, 123.1, 122.6,
110.8, 110.0, 56.1, 56.1
(E)-1-(2, 4-dihydroxyphenyl)-3-(3-((E)-3-(2-hydroxy-4-methoxyphenyl)-3-oxoprop-1-
en-1-yl)phenyl) prop-2-en-1-one (CYB-15)
Scheme 3. The structure of CYB-15
Yield 17.3%, mp 232~234℃. IR 3387, 3225, 2921, 2848, 2693, 1639, 1573, 1513, 1439,
1359, 1313, 1282, 1226, 1174, 1131, 1021, 974, 959, 851, 832,783, 750, 736, 691, 664,
629, 584, 560, 524, 504, 482, 450. 1H NMR (500 MHz, DMSO) δ 13.43 (s, 1H), 13.39 (s,
1H), 10.79 (s, 1H), 8.48 (s, 1H), 8.33 (d, J = 9.1 Hz, 1H), 8.25 (d, J = 9.0 Hz, 1H), 8.15-
8.08(m, 2H) 8.00 (t, J = 7.0 Hz, 2H), 7.91-7.85 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 6.62
(dd, J = 9.0, 2.0 Hz, 1H), 6.55 (d, J = 2.5 Hz, 1H), 6.46 (dd, J = 9.0, 2.5 Hz, 1H), 6.32 (d,
J = 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 191.8, 191.4, 166.2, 165.9, 165.8, 165.4,
143.4, 142.9, 135.4, 135.3, 133.2, 132.8, 131.1, 131.1, 129.5, 129.3, 122.1, 122.1, 113.9,
113.0, 108.3, 107.6, 102.6, 101.0, 55.8; HRMS m/z 415.11860 (calcd for C25H19O6,
415.11871)
3.2 The effects of chalcones on the ABCG2-mediated MDR cell lines
9

The cytotoxicity of the chalcones and bis-chalcones towards S1 and S1-M1-80 cells are
shown in Table 1. S1-M1-80 is a MX-selected ABCG2-overexpressing cell line from
human colon cancer cell line S1 [31]. Thus, we determined the concentration at which
chalcones and bis-chalcones exhibit less than 20% inhibition on the proliferation of S1
and S1-M1-80 cells (Table 1). From Figure 1, MX, at a concentration of 2.5 µM,
showed only 13% inhibition on the proliferation of S1-M1-80 cells. However, the
cytotoxicity of MX increased when combined with some chalcone derivatives. the
chalcones or bis-chalcones CYB-2, CYB-3, CYB-7, CYB-10, CYB-13 with four
methoxy groups, and CYB-15 with one methoxy group and three hydroxy groups showed
more than 30% inhibition on the ABCG2-mediated MDR. When MX was combined with
chalcone CYB-2, the proliferation of S1-M1-80 cells was inhibited by 65%, which was
higher than its demethylation product, CYB-4, which had 24% inhibition. When in
combination with bis-chalcone CYB-13, MX inhibited the proliferation of S1-M1-80
cells by 44%, which was higher than CYB-14 and CYB-15 with 13% and 33%
inhibition, respectively. In addition, CYB-15 with one methoxy group showed higher
inhibitory effect than CYB-14 without a methoxy group. Similarly, methoxy bis-chalcone
CYB-16 showed lower inhibitory effect than the corresponding hydroxy bis-chalcone
CYB-17 on the proliferation of S1-M1-80 cells. The position of the methoxy groups on
chalcones and the meta or para position of α, β-unsaturated carbonyl groups on bis-
chalcones have effects on the reversal activities of chalcones derivatives. 3, 4, 3’, 4’-
methoxy chalcone CYB-3 has stronger inhibition on ABCG2 than 2, 4, 2’, 4’-methoxy
chalcone CYB-5. Bis-chalcones CYB-7 and CYB-10 at meta position of the α, β-
10

unsaturated carbonyl groups showed stronger inhibitory activity than CYB-8 and CYB-
12 at para position, respectively. Among all the compounds, methoxy chalcones CYB-2
and CYB-3 exhibited the most potent inhibitory effect on the proliferation of S1-M1-80
cells with survival rates of 65% and 66%, respectively, while methoxy bis-chalcone
CYB-7 also showed effective reversal activity with 44% inhibition. The results indicated
that chalcones exhibit better reversal activities than the bis-chalcones. Most of the bis-
chalcones did not show significant reversal effects on ABCG2-mediated MDR. In the
previous report, the bis-chalcone showed a synergistic effect with an ABCG2 inhibitor,
chromones, to strength the inhibition of ABCG2 transporter [25]. Nevertheless, our study
showed that chalcones are more potent than the bis-chalcones, thus the extended π system
might not be promising to increase the reversal activities of chalcone derivatives. There
were similar findings in the investigation of indole derivatives as reversal agents for
ABCG2-mediated MDR [35]. Since the two compounds CYB-2 and CYB-3 showed
potent reversal effects on ABCG2-mediated MDR, the two compounds were selected for
further study. As shown in Figure 2, when combined with CYB-2 and CYB-3 at a
concentration of 5 μM, the IC50 values of MX on S1-M1-80 cells were 1.86 ± 0.10 and
3.29 ± 0.15 μM, respectively, indicating that CYB-2 is more potent than CYB-3. Thus,
CYB-2 was used for further mechanistic study. Fumitremorgin C (FTC), a well-known
ABCG2 inhibitor, was used as a positive control inhibitor.
Table 1. The cytotoxicity of chalcones and bis-chalcones, and the selected concentrations
for reversal study.
S1
S1-M1-80
Compd.
Structure
11

95%
95%
Concentration
for reversal
study c / μM
a
a
IC50
μM
/
IC50
μM
/
confidence
confidence
intervals b
intervals b
40.6±1.9
27.9±1.0
16.9±0.1
34.5±4.3
20.4 ~ 54.6 42.3±6.3
18.5~36.3 18.0±1.6
11.9 ~ 18.6 19.1±4.0
28.2~58.3
12.8 ~ 33.0
18.5 ~ 22.9
NA d
5
CYB-1
CYB-2
CYB-3
CYB-4
5
5
26.7 ~ 40.6
>100
10
36.3±6.7
>100
23.9 ~ 46.2 29.9±4.8
17.6~36.8
NA d
5
10
1
CYB-5
CYB-6
CYB-7
NA d
>100
3.2±0.8
2.2 ~ 3.6
4.0±0.5
2.9 ~ 4.6
>100
NA d
>100
>100
NA d
NA d
10
5
CYB-8
CYB-9
24.5±6.9
18.6 ~ 32.5
24.9±2.4
11.0±0.3
20.3 ~ 38.8 30.0±4.9
9.2 ~ 13.6 35.7±2.1
19.2 ~ 31.5
28.1 ~ 41.6
10
5
CYB-10
CYB-11
83.9±7.6
50.4 ~ 96.3
>100
NA d
10
CYB-12
12

2.7±0.9
2.5±0.4
2.1 ~3.3
2.0 ~ 3.4
7.1 ~ 9.2
NA d
3.5±0.9
4.5±0.2
8.6±0.8
>100
2.8 ~ 4.5
3.5 ~ 5.1
7.4 ~9.6
NA d
1
1
CYB-13
CYB-14
CYB-15
CYB-16
CYB-17
CYB-18
CYB-19
8.4±0.07
98.0±0.6
83.2±3.4
33.5±8.2
13.7±0.1
1
10
10
10
5
57.6 ~ 98.8
>100
NA d
29.2 ~40.5 39.1±1.8
11.5 ~ 14.5 19.1±1.8
31.6 ~ 48.2
11.3 ~ 20.4
a IC50 values were obtained by MTT assay, indicating the concentration at which the
compounds inhibited the cell proliferation by 50%. b 95% confidence intervals for IC50
values. c The concentration used for reversal study showed less than 20% inhibition on
the proliferation of S1 and S1-M1-80 cells. d The highest concentration used for
cytotoxicity assay was 100 μM. When IC50 value is closed to or higher than 100 μM, the
95% confidence intervals for IC50 value can not be obtained.
13

Figure 1. The inhibition of S1-M1-80 cell proliferation by the combination of the
chalcone derivatives and MX (2.5 μM). The inhibition of the proliferation was conducted
with MTT assay. The statistical significances between the treatment groups and the
control group were indicated as follows: *, P < 0.05, **, P < 0.01, ****, P < 0.0001, one-
way ANOVA.
Figure 2. The reversal effect of the chalcone derivatives CYB-2 and CYB-3 on S1-M1-
80 cells. IC50 values were obtained by MTT assay, indicating the concentration at which
14

the compounds inhibited the cell proliferation by 50%. The statistical significances
between the treatment groups and the control group were indicated as follow: ****, P <
0.0001, two-way ANOVA. #, P < 0.0001, indicating the statistical significances between
the multidrug resistant cell control groups and the parental cell groups, two-way
ANOVA.
3.3 The effects of CYB-2 on the ABCG2- and ABCB1--mediated MDR cell lines
The representative cytotoxic curves of CYB-2 on the cell lines that were used for
reversal activity study are shown in Figure 3, which indicates that CYB-2 exhibits less
than 20% inhibition on the proliferation of the cell lines at the tested concentration. As
shown in Figure 4A, 4B, CYB-2 significantly increased the cytotoxicity of MX in
ABCG2-overexpressing S1-M1-80 and NCI-H460/MX20 cell lines, and effectively
increased the cytotoxicity of doxorubicin in S1-M1-80 cell line (Figure 4C). Moreover,
CYB-2 enhanced the cytotoxicity of colchicine in ABCB1-overexpressing KB-C2 cells
(Figure 4D). However, CYB-2 cannot sensitize NCI-H460/MX20 or KB-C2 cells to
cisplatin, a non-substrate of the ABCG2 or ABCB1 (Figure 4E, 4F). In addition, CYB-2
can effectively reverse MDR in HEK/ABCG2-482-R2 and HEK/ABCB1 (Figure 4G,
4H), which were transfected with ABCG2 or ABCB1 using human embryonic kidney
293 HEK293/pcDNA3.1 cells. Thus, we concluded that CYB-2 sensitized both ABCG2-
and ABCB1-mediated MDR cell lines to anticancer drugs by interacting with ABCG2
and ABCB1.
15

Figure 3. The cytotoxic curves of CYB-2 on different cell lines.
16

Figure 4. Reversal effect of CYB-2 on ABCG2- and ABCB1- mediated MDR cell lines.
IC50 values were obtained by MTT assay, indicating the concentration at which the
compounds inhibited the cell proliferation by 50%. FTC and verapamil are used as
17

positive control inhibitors for ABCG2 and ABCB1, respectively. The statistical
significances between the treatment groups and the control group were indicated as
follows: ***, P < 0.001; ****, P < 0.0001, two-way ANOVA. #, P < 0.0001, indicating
the statistical significances between the multidrug resistant cell control groups and the
parental cell groups, two-way ANOVA.
Table 2. The different cell lines used in this study.
Parental /
multidrug resistant
ABC transporters
overexpressed
Cell lines
S1
Cell type
Human colon cancer
Human colon cancer
Human lung cancer
Human lung cancer
Parental
Multidrug resistant
Parental
/
S1-M1-80
NCI-H460
NCI-H460-MX20
KB-3-1
ABCG2
/
Multidrug resistant
Parental
ABCG2
/
Human cervical
carcinoma
Human cervical
carcinoma
KB-C2
Multidrug resistant
Parental
ABCB1
/
Human embryonic
kidney 293 cell line
HEK293/pcDNA3.1
HEK/ABCG2-482-R2
HEK/ABCB1
Human embryonic
kidney 293 cell line
Multidrug resistant
Multidrug resistant
ABCG2
ABCB1
Human embryonic
kidney 293 cell line
3.4 The effects of CYB-2 on the anticancer drugs accumulation and efflux in
ABCG2- and ABCB1-mediated MDR cell lines
From the accumulation and efflux assay, the results indicated that CYB-2 can
effectively increase the accumulation of [3H]-MX in ABCG2-mediated NCI-H460/MX20
cells (Figure 5A), and it is comparable with fumitremorgin C (FTC). CYB-2 can also
significantly increase the accumulation of [3H]-paclitaxel in the ABCB1-mediated KB-
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C2 cells (Figure 5B). CYB-2 did not change the efflux function of the parental cells
NCI-H460 and KB-3-1 (Figure 5C, 5D), while the efflux of anticancer drugs decreased
in multidrug resistant NCI-H460/MX20 and KB-C2 cell lines (Figure 5E, 5F). The
results revealed that CYB-2 increased the accumulation of anticancer drugs in NCI-
H460/MX20 and KB-C2, resulting from the inhibition of drug efflux, thus re-sensitizing
the MDR cell lines to anticancer drugs.
Figure 5. The effects of CYB-2 on the accumulation and efflux of [3H]-MX and [3H]-
paclitaxel in ABCG2- and ABCB1-mediated MDR cell lines. FTC was used as a positive
19

control inhibitor of ABCG2 and verapamil was used as a positive control inhibitor of
ABCB1. The statistical significances between the treatment groups and the control group
were indicated as follow: ****, P < 0.0001, two-way ANOVA.
3.5 The effects of CYB-2 on the expression and subcellular localization of ABCG2
and ABCB1 in MDR cells
The down-regulation of ABC transporters or translocation of ABC transporters from
membrane to cytoplasm can result in the decrease of MDR in cancer cells. The
immunoblot results showed that the expression of ABCG2 and ABCB1 did not
significantly change after the treatment of CYB-2 (Figure 6A, 6B). Moreover, the
subcellular localization of ABCG2 in NCI-H460/MX20 and ABCB1 in KB-C2 did not
alter after the treatment with CYB-2 (Figure 6C, 6D). Thus, we hypothesized that ABC
transporter activities were inhibited by CYB-2, which further inhibited the efflux
function of the ABC transporters.
20

Figure 6. (A) Immunoblot analysis showing the expression of ABCG2 in NCI-
H460/MX20 cells, and (B) ABCB1 in KB-C2 cells after the treatment with CYB-2. The
effects of CYB-2 on subcellular localization of (C) ABCG2 and (D) ABCB1 in MDR
cells.
3.6 The effect of CYB-2 on ABCG2 and ABCB1 ATPase activities
Previous reports have shown that both the inhibition and stimulation of ABC
transporters ATPase can result in the reverse of MDR [42,43]. Thus, CYB-2 blocks the
21

efflux of anticancer drugs due to inhibition of ABCG2 or ABCB1 ATPase activities, or
stimulates the ATPase activity and works as a competitive substrate. To determine
whether the activities of ABCG2 and ABCB1 ATPase were inhibited or stimulated, we
performed the ATPase assay. The results (Figure 7) suggested that CYB-2 inhibited the
activities of ATPase in both ABCG2 and ABCB1 with IC50 values of 0.95 µM and 2.01
µM, respectively. CYB-2 exhibits strong inhibition on ABCG2 and ABCB1 ATPase,
thus blocking the efflux function of MDR cancer cells and increasing the accumulation of
anticancer drugs.
Figure 7. The effects of CYB-2 on the accumulation and efflux function of [3H]-MX and
[3H]-paclitaxel on ABCG2- and ABCB1-mediated MDR cell lines. IC50 values indicate
the concentration at which CYB-2 inhibit the cell proliferation by 50%.
3.7 The binding mode of CYB-2 with ABCG2 and ABCB1
The docked positions of CYB-2 within the binding cavity of human ABCG2 (PDB:
6FFC) (Figure 8A) and ABCB1 (PDB: 6FN1) (Figure 8D) are shown in Figure 8 with
docking scores -9.607 kcal/mol and -8.028 kcal/mol, respectively. One phenyl ring of
22

CYB-2 forms a π-π interaction with Phe439 in the B chain of the ABCG2 protein
(Figure 8B). Apart from the π-π interaction, CYB-2 also has interactions with the
residues Leu405, Phe432, Val546, and Met549 of ABCG2 (Figure 8C). For the
interaction between CYB-2 and ABCB1 protein, the residue Tyr982 forms π-π stacking
with one phenyl ring of CYB-2. In the meantime, a methoxy group forms a hydrogen
bond with Gln945 (-OCH3•••H2N-Gln945), and the carbonyl group of CYB-2 has a
hydrogen binding with Tyr952 (Figure 8E). Moreover, CYB-2 interacts with a
hydrophilic pocket of ABCB1 with residues such as Leu64, Met67, Met68, Phe727,
Phe731, Met948, Tyr949, Tyr952, Phe977, and Phe982 (Figure 8F).
Figure 8. Docked positions of CYB-2 with the human ABCG2 and ABCB1 transporter proteins.
(A) CYB-2 in the binding site of ABCG2 indicated with a black circle. (B) The binding of CYB-2
and the human ABCG2 transporter protein in a ball-and-stick model. CYB-2 is shown with
23

following color code: carbon - cyan, hydrogen - white, and oxygen - red. The important interaction
was indicated: ring centroids - blue dotted line. (C) The interactions of CYB-2 with human
ABCG2. The interaction was indicated as follows: π-π stacking interaction - the green line. (D)
CYB-2 in the binding site of ABCB1 indicated with a black circle. (E) The binding of CYB-2 and
the human ABCB1 protein in a ball-and-stick model. The important interactions were presented:
ring centroids - blue dotted lines; hydrogen bonds - dotted yellow lines. (F) The interactions of
CYB-2 with the binding site of the human ABCB1 protein. The interactions were indicated as
follows: hydrogen bonds - purple arrow; π-π stacking aromatic interactions - the green lines.
In conclusion, this study investigated the effect of methoxy and hydroxy substitutions and
the extended π system on the potencies of chalcones and bis-chalcones. From the
structure-activity relationship study, the methoxy chalcones or bis-chalcones showed
potent inhibition on ABCG2-mediated MDR, and demonstrated that methoxy groups
enhance the reversal activities of chalcones. Moreover, the methoxy chalcones CYB-2
and CYB-3 exhibit more potent reversal activities than methoxy bis-chalcones, indicating
that the extended π system does not enhance the potency of the chalcone derivatives. In
addition, the position of the methoxy groups of chalcones and the α, β-unsaturated
carbonyl groups of bis-chalcones have effects on the inhibition of ABCG2. Among all the
compounds, methoxy chalcone CYB-2 showed the best reversal activity on ABCG2-
mediated MDR; in addition, it can also reverse ABCB1-mediated MDR. The mechanistic
studies indicated that CYB-2 inhibits the basal ATP hydrolysis of both ABCG2 and
ABCB1 transporters, thereby inhibiting the efflux function of the transporters and thus
increasing the intracellular accumulation of certain substrates into the MDR cells without
affecting ABCG2 or ABCB1 protein expression and localization. The previous report
indicates that bis-chalcone can reverse ABCG2-mediated MDR [25], in addition, some
24

non-basic chalcones can also selectively reverse ABCG2-mediated MDR [26,27]. In this
study, non-basic chalcone CYB-2 show stronger reversal activity on ABCG2 than all the
bis-chalcones we tested in this study. CYB-2 also exhibits inhibitory activity on ABCB1,
which is a superiority compared to the reported non-basic chalcones with specific
ABCG2 inhibition in the treatment of ABCG2 and ABCB1 co-expressing cancers.
Furthermore, as ABCG2/ABCB1 duel inhibitors, non-basic chalcones have synthetic
tractability compared to the reported chalcone-based derivatives, such as bifendate-
chalcone hybrids and flavone-based chalcones. These results revealed that non-basic
methoxy chalcones have potential to be developed as reversal agents to overcome MDR
in ABCG2 and ABCB1 co-expressing cancers.
Author information
*Zhe-Sheng Chen: e-mail, chenz@stjohns.edu; phone, 1-718-990-1432
*Bo Wang: e-mail, ceswb@mail.sysu.edu.cn; phone, +86-84113083
*Cai-Ping Tan: e-mail, tancaip@mail.sysu.edu.cn; +86-20-84113780
Acknowledgments
We thank Dr. Susan E. Bates and Dr. Robert W. Robey (NCI, NIH, MD) for generously
providing us the NCI-H460, NCI-H460/MX20, HEK293/pcDNA3.1, and transfected
HEK/ABCG2-482-R2 cell lines. We thank Dr. Shin-ichi Akiyama (Kagoshima
University, Kagoshima, Japan) for kindly providing us the KB-3-1 and KB-C2 cell lines.
We are thankful to Dr. Tanaji T. Talele (St. John’s University, Queens, NY) providing us
the support for the docking study. We thank Juan-Juan Huang (Technical University of
Munich, Garching, Germany) for editing the graphic abstract. This work was supported
25

by the Department of Pharmaceutical Science at St. John’s University and the National
Natural Science Foundation of China (21272290, J1103305).
Conflicts of interest
The authors declared no conflicts of interest.
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