Chalcones with electron-withdrawing and electron-donating substituents: Anticancer activity against TRAIL resistant cancer cells, structure-activity relationship analysis and regulation of apoptotic proteins

Article abstract of DOI:10.1016/j.ejmech.2014.03.002

In the present study, a series of 46 chalcones were synthesised and evaluated for antiproliferative activities against the human TRAIL-resistant breast (MCF-7, MDA-MB-231), cervical (HeLa), ovarian (Caov-3), lung (A549), liver (HepG2), colorectal (HT-29), nasopharyngeal (CNE-1), erythromyeloblastoid (K-562) and T-lymphoblastoid (CEM-SS) cancer cells. The chalcone 38 containing an amino (-NH₂) group on ring A was the most potent and selective against cancer cells. The effects of the chalcone 38 on regulation of 43 apoptosis-related markers in HT-29 cells were determined. The results showed that 20 apoptotic markers (Bad, Bax, Bcl-2, Bcl-w, Bid, Bim, CD40, Fas, HSP27, IGF-1, IGFBP-4, IGFBP-5, Livin, p21, Survivin, sTNF-R2, TRAIL-R2, XIAP, caspase-3 and caspase-8) were either up regulated or down regulated.

Full text of DOI:10.1016/j.ejmech.2014.03.002

European Journal of Medicinal Chemistry 77 (2014) 378e387
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chalcones with electron-withdrawing and electron-donating
substituents: Anticancer activity against TRAIL resistant cancer cells,
structureeactivity relationship analysis and regulation of apoptotic
proteins
Chun Wai Mai a, Marzieh Yaeghoobi b, Noorsaadah Abd-Rahman b, Yew Beng Kang a,
,
Mallikarjuna Rao Pichika a
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala
Lumpur, Malaysia
b Drug Design and Development Research Group, Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a series of 46 chalcones were synthesised and evaluated for antiproliferative activities
against the human TRAIL-resistant breast (MCF-7, MDA-MB-231), cervical (HeLa), ovarian (Caov-3), lung
(A549), liver (HepG2), colorectal (HT-29), nasopharyngeal (CNE-1), erythromyeloblastoid (K-562) and
T-lymphoblastoid (CEM-SS) cancer cells. The chalcone 38 containing an amino (-NH2) group on ring A was
the most potent and selective against cancer cells. The effects of the chalcone 38 on regulation of 43
apoptosis-related markers in HT-29 cells were determined. The results showed that 20 apoptotic markers
(Bad, Bax, Bcl-2, Bcl-w, Bid, Bim, CD40, Fas, HSP27, IGF-1, IGFBP-4, IGFBP-5, Livin, p21, Survivin, sTNF-R2,
TRAIL-R2, XIAP, caspase-3 and caspase-8) were either up regulated or down regulated.
Received 10 November 2013
Received in revised form
28 February 2014
Accepted 2 March 2014
Available online 3 March 2014
Keywords:
Chalcones
Anti-proliferative
Ó 2014 Elsevier Masson SAS. All rights reserved.
Apoptosis proteins
TRAIL-resistant tumours
Human colorectal cancer (HT-29) cells
1. Introduction
carbonyl system could hinder their biological activities [2]. A
number of synthetic modifications, viz., such as oxathiolone fused
Worldwide, cancer is one of the leading causes of death. Patients
diagnosed with cancer often experience a poor quality of life due to
the adverse events associated with cancer. Chemotherapy is one of
the effective approaches in suppressing tumour growth and erad-
ication of tumours. However, many patients undergoing chemo-
therapy suffer from associated side effects such as nausea,
vomiting, cachexia, lethargy and poor oral intake. Although, many
research studies have reported potential chemotherapeutic effects
of novel compounds, the search for new anti-cancer agents with
improved efficacy and reduced side effects continues. [1]
[3], boron substituted [4], heterocyclic infused [5], biphenyl based
[6], imidazolones linked [7], coumarin based chalcones [8] or other
substitutions [9e13]; have also been reported to affect the biolog-
ical activities including anticancer activities of chalcones.
Tumour necrosis factor (TNF)-related apoptosis-inducing ligand
(TRAIL) is an attractive target in cancer research because it is
capable of selectively inducing apoptosis in cancer cells without
affecting normal cells [14]. Five receptors, such as TRAIL-R1 (DR4,
death receptor 4), TRAIL-R2 (DR5), DR6, TRAILR-3 (decoy receptor
(DcR)1) and TRAIL-R4 (DcR2); have been identified for TRIAL. Two
of these receptors DR4 and DR5 have cytoplasmic death domains
and trigger TRAIL induced apoptosis [15]. Other receptors, DcR1
and DcR2, are expressed on the cell surface and protect the cancer
cells from TRAIL induced apoptosis [16]. The last TRAIL receptor,
osteoprotegerin, is a less studied receptor and is known to inhibit
the tumouricidal activity of TRAIL [17]. Interaction of TRAIL with
DR4 and DR5 results in caspase-8 activation, which induces
apoptosis by activating either caspase-3 or the intrinsic
mitochondria-mediated apoptotic pathway [18]. However, TRAIL
Chalcones have attracted much attention due to their diverse
biological activities, such as anti-cancer, anti-oxidant, anti-
inflammatory, and/or anti-infective activities. Chalcones consist of
two aromatic rings connected by an
a,b-unsaturated carbonyl
group. It has been shown that the removal of
a,b-unsaturated
* Corresponding author.
E-mail address: mallikarjunarao_pichika@imu.edu.my (M.R. Pichika).
http://dx.doi.org/10.1016/j.ejmech.2014.03.002
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
379
Table 1
Chalcone derivatives and their Lipinski’s rule of five parameters.
Chalcone
Ring A
Ring B
Lipinski rule of 5 parameters
No.
1
2
3
4
5
6
7
8
R2
H
H
H
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
R4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R6
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
R20
H
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
R30
H
H
OH
H
H
H
OH
H
H
R40
H
H
H
OH
H
H
R50
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
R60
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
MW
A log P
3.70
3.46
3.46
3.46
3.46
3.22
3.22
3.22
3.44
3.43
3.41
3.23
3.95
4.12
4.37
3.59
3.41
4.18
3.43
3.32
3.32
3.56
4.33
4.42
3.44
3.44
3.62
4.33
4.21
3.89
3.67
4.35
4.59
4.35
4.39
4.85
4.73
2.96
2.85
3.62
2.94
3.86
2.96
3.62
3.62
4.28
HBA
1
2
2
2
2
3
3
3
3
4
5
4
2
2
2
5
5
4
4
6
6
5
3
3
5
5
6
3
4
2
3
2
2
2
1
1
2
2
4
2
3
2
2
2
2
2
HBD
208.26
224.26
224.26
224.26
224.26
240.25
240.25
240.25
254.28
284.31
314.33
268.26
238.28
258.70
274.31
327.37
314.33
363.20
284.31
329.30
329.30
313.31
302.75
347.20
312.32
312.32
358.39
302.75
314.40
256.27
268.31
272.73
288.34
265.35
240.27
256.73
268.37
223.27
268.27
257.72
253.30
273.33
223.27
257.72
257.72
292.16
0
1
1
1
1
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
OCH3
H
H
H
H
H
H
H
NH2
NH2
NH2
NH2
NH2
H
H
H
H
H
OH
OCH3
OCH3
OCH3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
OCH3
OCH3
1,3-dioxolane
H
H
CH3
Cl
Phenyl
H
H
H
H
H
H
H
H
H
N(CH3)2
OCH3
Br
H
H
NO2
NO2
Cl
Br
H
1,3-dioxolane
1,3-dioxolane
OCH3
H
H
H
H
H
Phenyl
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
Cl
SCH3
F
H
OCH3
OCH3
H
OCH3
OCH3
OCH3
CH3
CH3
CH3
CH3
H
H
H
H
H
Cl
N(CH3)2
F
Cl
SMe
H
NO2
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
OCH3
Phenyl
NH2
NH2
NH2
NH2
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
MW: Molecular Weight; A log P: logarithm of octanolewater partition coefficient; HBA: number of hydrogen bond acceptor; HBD: number of hydrogen bond donor.
resistance in cancer cells have been reported elsewhere in the
literature [19e21]. Few recent studies have reported the potential
of chalcones in inducing apoptosis in TRAIL-resistant cancer cells
[22,23].
activity relationships. Regulation of the various apoptotic proteins
in the most sensitive cancer cell, HT-29, by the most potent chal-
cone 38, was also determined.
In the current study, we have synthesised 46 chalcones con-
sisting wide range of electron-withdrawing and electron-donating
substituents that obey Lipinski’s rule of five. The apoptosis inducing
capability of these chalcones in 10 TRAIL-resistant cancer cells and
selective toxicity towards cancer cells compared to normal cells
was determined with an aim to derive the important structure
2. Results and discussion
2.1. Chemistry
The chalcones were classified into 12 groups according to their
substitution patterns on ring A. Group 1 chalcones (1e4) have no

Table 2
The anti-proliferative effects of chalcones over cancer cells.
Chalcone No.
MCF-7 IC50
(
m
M)
MDA-MB-231 IC50
(
m
M)
HeLa IC50
(m
M)
Caov-3 IC50
(m
M)
A549 IC50
(m
M)
HepG2 IC50
(m
M)
HT-29 IC50
(
m
M)
CNE-1 IC50
(m
M)
K562 IC50
(
m
M)
CEM-SS IC50 (mM)
1
2
3
4
5
6
7
8
>100
>100
>100
>100
69.79 ꢀ 3.15
65.71 ꢀ 1.70
31.43 ꢀ 1.97
77.76 ꢀ 2.63
47.78 ꢀ 1.20
24.04 ꢀ 0.53
56.36 ꢀ 1.76
67.61 ꢀ 5.88
61.68 ꢀ 0.36
40.41 ꢀ 0.98
20.97 ꢀ 0.14
23.27 ꢀ 0.66
33.50 ꢀ 1.02
34.038 ꢀ 7.67
15.26 ꢀ 6.03
>100
76.87 ꢀ 4.92
16.61 ꢀ 0.68
8.05 ꢀ 1.40
15.04 ꢀ 5.70
11.24 ꢀ 0.03
86.12 ꢀ 1.38
61.53 ꢀ 1.41
37.06 ꢀ 4.16
74.38 ꢀ 5.56
17.89 ꢀ 1.13
5.52 ꢀ 0.11
93.20 ꢀ 0.96
95.03 ꢀ 0.63
66.70 ꢀ 2.04
45.67 ꢀ 4.81
92.32 ꢀ 0.48
>100
>100
>100
>100
>100
>100
26.96 ꢀ 1.26
16.69 ꢀ 1.05
>100
76.81 ꢀ 3.21
47.16 ꢀ 1.56
36.44 ꢀ 2.16
>100
25.98 ꢀ 7.79
10.01 ꢀ 2.54
32.26 ꢀ 0.34
38.41 ꢀ 1.73
16.22 ꢀ 2.98
16.32 ꢀ 2.98
43.29 ꢀ 0.06
41.89 ꢀ 0.50
18.33 ꢀ 3.67
20.22 ꢀ 3.08
23.06 ꢀ 0.68
62.08 ꢀ 2.00
21.90 ꢀ 1.59
>100
45.05 ꢀ 0.45
61.87 ꢀ 0.20
63.72 ꢀ 0.46
77.29 ꢀ 0.38
32.26 ꢀ 0.48
19.84 ꢀ 0.29
87.54 ꢀ 1.01
>100
>100
>100
85.81 ꢀ 6.00
81.92 ꢀ 4.35
41.50 ꢀ 3.41
>100
37.21 ꢀ 0.90
19.63 ꢀ 0.34
36.75 ꢀ 1.80
>100
32.30 ꢀ 1.67
20.59 ꢀ 0.94
57.96 ꢀ 4.91
42.65 ꢀ 2.92
75.71 ꢀ 2.20
27.21 ꢀ 5.11
68.35 ꢀ 6.86
>100
>100
>100
>100
>100
27.81 ꢀ 1.17
11.74 ꢀ 1.76
>100
32.19 ꢀ 1.64
21.35 ꢀ 0.63
63.33 ꢀ 2.35
90.74 ꢀ 2.70
47.72 ꢀ 1.93
42.78 ꢀ 3.15
94.64 ꢀ 2.97
>100
58.11 ꢀ 2.72
18.52 ꢀ 3.91
33.24 ꢀ 2.49
69.15 ꢀ 2.12
>100
>100
>100
>100
36.43 ꢀ 1.87
14.37 ꢀ 4.44
>100
9.95 ꢀ 1.12
78.41 ꢀ 0.70
29.84 ꢀ 0.66
46.40 ꢀ 1.37
75.12 ꢀ 1.86
37.96 ꢀ 2.02
40.20 ꢀ 2.00
71.00 ꢀ 3.05
>100
16.76 ꢀ 4.51
10.06 ꢀ 1.89
28.30 ꢀ 1.21
18.40 ꢀ 0.73
>100
36.07 ꢀ 0.41
34.22 ꢀ 0.96
19.24 ꢀ 0.20
18.00 ꢀ 2.42
39.39 ꢀ 1.45
32.75 ꢀ 1.30
38.56 ꢀ 1.27
36.01 ꢀ 1.01
18.91 ꢀ 0.80
23.65 ꢀ 1.12
23.28 ꢀ 0.10
20.81 ꢀ 1.26
22.36 ꢀ 3.09
22.96 ꢀ 2.44
>100
7.94 ꢀ 0.47
5.86 ꢀ 0.10
19.33 ꢀ 3.41
76.45 ꢀ 3.22
15.97 ꢀ 1.57
20.75 ꢀ 2.90
35.20 ꢀ 3.72
63.81 ꢀ 2.29
25.22 ꢀ 2.16
22.85 ꢀ 1.78
35.95 ꢀ 5.01
66.00 ꢀ 3.69
55.99 ꢀ 9.48
21.66 ꢀ 1.91
>100
14.36 ꢀ 2.05
5.81 ꢀ 0.24
70.75 ꢀ 10.08
45.25 ꢀ 5.92
32.74 ꢀ 4.81
56.25 ꢀ 2.45
>100
9
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
63.90 ꢀ 1.95
12.00 ꢀ 1.96
16.01 ꢀ 1.27
20.96 ꢀ 3.02
27.87 ꢀ 4.83
31.86 ꢀ 3.25
>100
32.05 ꢀ 11.00
17.68 ꢀ 5.04
22.84 ꢀ 4.92
55.61 ꢀ 3.39
48.31 ꢀ 0.93
>100
>100
>100
13.67 ꢀ 1.42
6.61 ꢀ 0.72
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
66.38 ꢀ 7.24
10.22 ꢀ 0.42
11.43 ꢀ 0.19
66.72 ꢀ 1.82
16.16 ꢀ 4.02
31.19 ꢀ 3.70
24.00 ꢀ 5.13
10.89 ꢀ 0.18
>100
36.28 ꢀ 7.90
36.68 ꢀ 0.66
43.65 ꢀ 0.49
>100
>100
>100
14.04 ꢀ 1.50
23.02 ꢀ 6.07
9.58 ꢀ 0.81
13.45 ꢀ 1.82
>100
18.17 ꢀ 6.06
28.26 ꢀ 6.87
>100
82.25 ꢀ 6.54
19.00 ꢀ 1.16
>100
8.36 ꢀ 0.80
26.57 ꢀ 1.18
>100
13.59 ꢀ 2.02
4.96 ꢀ 0.04
21.00 ꢀ 0.86
>100
>100
>100
>100
>100
>100
>100
>100
>100
78.71 ꢀ 1.89
10.04 ꢀ 0.51
94.90 ꢀ 6.57
16.06 ꢀ 2.99
84.54 ꢀ 6.69
31.37 ꢀ 2.66
11.87 ꢀ 0.04
59.11 ꢀ 7.30
12.06 ꢀ 0.68
4.68 ꢀ 0.29
21.13 ꢀ 1.05
55.54 ꢀ 5.97
>100
21.26 ꢀ 0.88
9.77 ꢀ 1.34
88.23 ꢀ 4.20
7.72 ꢀ 0.66
47.00 ꢀ 2.96
30.04 ꢀ 4.48
27.47 ꢀ 1.99
78.31 ꢀ 3.96
8.71 ꢀ 0.74
5.38 ꢀ 0.41
36.22 ꢀ 2.58
72.97 ꢀ 4.45
>100
>100
43.32 ꢀ 2.61
11.72 ꢀ 0.72
8.60 ꢀ 0.11
7.66 ꢀ 1.72
>100
>100
>100
>100
>100
>100
>100
>100
79.53 ꢀ 3.63
69.35 ꢀ 1.37
>100
39.39 ꢀ 2.31
4.92 ꢀ 0.17
55.40 ꢀ 0.70
>100
82.09 ꢀ 2.28
29.45 ꢀ 2.18
>100
11.31 ꢀ 0.16
4.56 ꢀ 0.21
28.37 ꢀ 5.63
>100
5.00 ꢀ 0.26
17.61 ꢀ 3.29
>100
5.51 ꢀ 0.71
4.54 ꢀ 0.20
40.25 ꢀ 2.05
>100
>100
7.53 ꢀ 0.71
13.63 ꢀ 2.04
>100
>100
>100
14.84 ꢀ 2.17
22.18 ꢀ 0.58
>100
>100
>100
17.91 ꢀ 0.43
>100
>100
>100
>100
>100
>100
>100
>100
>100
36.59 ꢀ 1.61
77.51 ꢀ 1.04
>100
91.73 ꢀ 10.31
61.15 ꢀ 0.85
5.07 ꢀ 0.53
>100
>100
>100
>100
34.50 ꢀ 0.68
31.02 ꢀ 0.99
>100
>100
21.11 ꢀ 1.92
5.70 ꢀ 0.31
61.33 ꢀ 2.80
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
11.43 ꢀ 0.45
13.08 ꢀ 3.22
72.11 ꢀ 2.67
>100
>100
>100
>100
>100
4.39 ꢀ 0.25
5.36 ꢀ 0.17
5.93 ꢀ 1.56
>100
>100
>100
>100
>100
>100
>100
4.99 ꢀ 0.11
8.84 ꢀ 0.38
36.29 ꢀ 2.92
69.10 ꢀ 4.01
21.55 ꢀ 0.11
42.54 ꢀ 1.58
12.14 ꢀ 0.11
10.16 ꢀ 0.36
>100
>100
64.10 ꢀ 5.30
93.01 ꢀ 3.39
15.81 ꢀ 3.36
31.63 ꢀ 4.56
>100
10.29 ꢀ 3.30
15.11 ꢀ 3.12
36.62 ꢀ 6.86
>100
21.06 ꢀ 2.43
28.71 ꢀ 7.97
9.38 ꢀ 0.18
7.91 ꢀ 0.83
29.82 ꢀ 3.82
>100
72.83 ꢀ 7.29
>100
>100
>100
63.03 ꢀ 5.83
46.15 ꢀ 3.69
95.27 ꢀ 3.66
>100
>100
>100
>100
>100
>100
5.42 ꢀ 0.17
8.15 ꢀ 1.03
14.44 ꢀ 4.65
>100
44.35 ꢀ 2.34
15.58 ꢀ 0.88
26.45 ꢀ 6.41
>100
>100
>100
5.32 ꢀ 0.34
19.26 ꢀ 0.99
93.24 ꢀ 3.03
5.68 ꢀ 0.12
10.97 ꢀ 1.89
>100
78.13 ꢀ 9.90
15.37 ꢀ 0.13
69.16 ꢀ 2.64
12.30 ꢀ 2.25
28.52 ꢀ 4.60
94.08 ꢀ 3.71
8.27 ꢀ 4.69
45.90 ꢀ 3.17
>100
>100
>100
>100
>100
>100
>100
>100
35.58 ꢀ 1.23
42.84 ꢀ 3.20
20.68 ꢀ 0.54
12.50 ꢀ 0.51
>100
>100
>100
19.60 ꢀ 3.45
36.87 ꢀ 3.87
17.46 ꢀ 4.30
>100
72.97 ꢀ 3.63
23.46 ꢀ 1.34
18.46 ꢀ 3.88
>100
78.10 ꢀ 6.50
49.23 ꢀ 7.97
38.56 ꢀ 1.59
>100
14.93 ꢀ 1.15
19.13 ꢀ 4.67
>100
23.51 ꢀ 1.29
>100

C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
381
ring A substitution. Group 2 chalcones (5e15) have ring A
substituted with hydroxy group at position 2. Groups 3 and 4 are
variants of group 2 in which addition of two methoxy groups at
positions 4 and 6 resulted in group 3 (16e20) and at positions 3 and
4 gave rise to group 4 (21). Group 5 chalcones (30) have ring A
substituted with methoxy group at position 2. Groups 6 and 7 are
variants of group 5 in which addition of one methoxy at position 4
resulted in group 6 (22e25) and at position 5 gave group 7 (26e27).
Group 8 chalcones (31e33) have ring A substituted with methoxy
group at position 4. Groups 9 and 10 are variants of group 8 in
which an additional methoxy group appeared at position 3 in the
case for group 9 (28e29) and the methyl group is replaced by a
methoxy group at position 4 resulted in group 10 (34e37). Group 11
chalcones (38e42) had ring A substituted with amino group at
position 2. Group 12 chalcones (43e46) had ring A substituted with
amino group at position 4. The major substituents on ring B were
the halogens (F, Cl, Br), electron donating groups (OH, OCH3, SCH3,
1,3-dioxolane), or electron withdrawing groups (NO2). In some
chalcones (15, 33, 42) ring B was replaced with naphthalene ring.
All the chalcones synthesized in this study obey the Lipinski’s rule
of five (Table 1) suggesting these chalcones could be potential orally
active drug candidates [24].
2.2. Biological activity
2.2.1. Anti-proliferative effects of chalcones on cancer cells and its
structureeactivity relationships
Chalcones synthesised in this study were tested for their anti-
proliferative effects for 72 h at 37 ꢁC using the methyl thiazolyl
tetrazolium (MTT) assay on 10 different cancer cell lines, viz., hu-
man oestrogen receptor positive breast cancer cells (MCF7), human
oestrogen receptor negative breast cancer cells (MDA-MB-231),
human cervical cancer cells (HeLa), human ovarian cancer cells
(Caov-3), human lung cancer cells (A549), human liver cancer cells
(HepG2), human colorectal cancer cells (HT-29), human nasopha-
ryngeal cancer cells (CNE-1), human T-lymphoblastoid leukaemia
cells (CEM-SS), human erythromyeloblastoid leukaemia cells
(K562) and normal human embryonic kidney (HEK-293) cells
respectively.
Table 2 lists the IC50 values of the chalcones against human
cancer cell lines (MCF-7, MDA-MB-231, HeLa, Caov-3, A549, Hep G2,
HT-29, CNE-1, K562, CEM-SS) and Table 3 lists the IC50 values of
chalcones against normal human embryonic kidney (HEK-293)
cells. Therefore selectivity ratios (IC50 HEK-293/IC50 HT-29) of the
chalcones were calculated using the following formula:
Table 3
The IC50 values of chalcones over HEK-293 and HT-29 cells with corresponding
selectivity ratios.
Chalcone no.
HEK-293 IC50
(
mM)
HT-29 IC50
(
m
M)
Selectivity ratio
1
2
3
4
5
6
7
8
86.93 ꢀ 6.97
67.98 ꢀ 4.59
22.23 ꢀ 0.49
33.77 ꢀ 5.21
48.68 ꢀ 0.55
48.20 ꢀ 9.80
53.79 ꢀ 3.49
>100
22.93 ꢀ 0.278
71.47 ꢀ 8.92
37.88 ꢀ 1.80
45.66 ꢀ 3.83
46.30 ꢀ 1.77
42.75 ꢀ 2.70
73.40 ꢀ 4.70
>100
>100
>100
21.71 ꢀ 0.14
66.85 ꢀ 1.4
81.42 ꢀ 0.93
>100
>100
>100
>100
>100
<1
9.95 ꢀ 1.12
6.83
1.55
5.81
0.69
1.07
1.64
>1.78
<1
2.23
2.14
2.00
0.83
0.88
<1
IC50 of chalcones against HEK ꢂ 293 cells
Selectivity ratio ¼
14.36 ꢀ 2.05
5.81 ꢀ 0.24
IC50 of chalcones against HT ꢂ 29 cells
70.75 ꢀ 10.08
45.25 ꢀ 5.92
32.74 ꢀ 4.81
56.25 ꢀ 2.45
>100
The selectivity ratios of chalcones are shown in Table 3. Arbi-
trary thresholds of IC50 against HT-29 ꢃ 10
mM and selectivity
ratio ꢄ 5 (Table 3) were used to identify promising compounds.
Eight (chalcone 2, 4, 21, 23, 27, 32, 38 and 39) out of forty-six
chalcones fulfilled the threshold limits for IC50 and selectivity ra-
tio. The contribution of either the electron-withdrawing or
electron-donating substituents to anti-proliferative effect was
assessed by making the following comparisons.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
32.05 ꢀ 11.00
17.68 ꢀ 5.04
22.84 ꢀ 4.92
55.61 ꢀ 3.39
48.31 ꢀ 0.93
>100
>100
>100
13.67 ꢀ 1.42
6.61 ꢀ 0.72
>100
Firstly, we compared the activities (based on IC50 HT-29 values)
of the unsubstituted chalcone 1 with mono-hydroxy substituted
chalcones 2e5. The chalcones with eOH group at either ortho- or
para- positions on aromatic ring B were shown potent and selective
antiproliferative effect on HT-29 cells. The eOH groups on both
rings A and B of chalcones 6e8 did not improve the anticancer
activity compared to that of mono hydroxy substituted chalcones in
which eOH group is present on either A or B ring. Hydroxyl group
substitutions in chalcones have been shown to play a key role in
anti-cancer activities of chalcones [25]. These results suggest that
the presence of eOH group and its position on ring B are important
in determining the potency and selectivity.
Secondly, comparisons were made to determine if having
another substituent (eOCH3, e1,3-dioxolane, eCH3, eCl, eC6H5, e
N(CH3)2, eBr) in addition to eOH group (Chalcones 9e21) made a
difference to activity. There was no specific trend observed in
antiproliferative activity. Chalcone 21 containing eNO2 group at
para-position on ring B was the most potent and selective.
Thirdly, comparisons were made to determine if having more
n.d.
n.d.
>7.31
3.28
<1
10.63
n.d.
>20
>5.68
n.d.
4.17
6.94
2.21
n.d.
n.d.
<1
>17.54
n.d.
n.d.
n.d.
n.d.
n.d.
>22.78
6.77
>16.86
n.d.
<1
<1
7.66 ꢀ 1.72
>100
5.00 ꢀ 0.26
17.61 ꢀ 3.29
>100
5.51 ꢀ 0.71
4.54 ꢀ 0.20
40.25 ꢀ 2.05
>100
22.95 ꢀ 0.13
60.44 ꢀ 8.71
88.83 ꢀ 2.07
>100
>100
>100
>100
83.85 ꢀ 10.58
>100
5.70 ꢀ 0.31
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.39 ꢀ 0.25
5.36 ꢀ 0.17
5.93 ꢀ 1.56
>100
>100
>100
36.31 ꢀ 3.02
than one electron withdrawing/donating groups (-OCH3, eCH3 e
>100
NO2, eF, eCl, eBr, e1,3-dioxolane, eSCH3, eN(CH3)2, eC6H5) on
either ring (chalcones 22e37) made a difference in activity. There
was no specific trend and all the chalcones were neither potent nor
selective. Chalcones 23 and 32 with a substituent eCl on ring B and
chalcone 27 with three eOCH3 groups were potent and selective
>100
44.42 ꢀ 1.17
95.34 ꢀ 2.48
72.39 ꢀ 9.65
86.18 ꢀ 0.35
83.43 ꢀ 5.07
14.93 ꢀ 1.15
19.13 ꢀ 4.67
>100
4.84
4.50
<1
indicating that the substituents eCl and eOCH3 on ring
B
n.d. ¼ cannot be quantified.
contribute to the antiproliferative activity of chalcones.

382
C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
Fig. 1. (A) The dose response curve of the chalcone 38 against HEK-293 and HT-29 cells, (B) morphological changes in HEK-293 and HT-29 cells upon treatment with chalcone 38.
Fourthly, comparisons were made to determine the effect of e
NH2 group on ring A and other substituent (eNO2, eCl, eOCH3, e
C6H5) on ring B (chalcones 38e46) made a difference in activity.
Chalcones 38 and 42 showed the most potent anti-proliferative
effect against HT-29 cells indicating that the eNH2 group on ring
A plays an important role.
Among all those tested, chalcone 38 exhibits the most potent
anti-proliferative effects with lowest IC50 (4.39
ꢀ
0.25 mM)
demonstrated against HT-29 cells. The dose response curves
(Fig.1A) showed that the chalcone 38 exhibited lower percentage of
cell viability in HT-29 cells at all concentrations when compared to
HEK-293 cells. The anti-proliferative effects exhibited by the chal-
cone 38 were also dose dependent. Observation under the micro-
scope (Fig.1B) showed no significant morphological change in HEK-
293 cells treated 1e10 mM of chalcone 38, as compared to HEK293
cells treated with negative control (1% dimethylsulfoxide, DMSO).
However we observed that the number of viable HT-29 cells was
reduced when HT-29 cells were exposed to 1e10 mM of chalcone
38. When compared to the negative control, treatment of chalcone
38 caused HT-29 cells to be shrunken and/or rounded in shape.
Some cells began to detach thereby loosing their cellecell and celle
plate interactions. The above observations were similar to the
Fig. 2. The mode of cell death induced by the selected chalcones in HT-29 cells.

C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
383
morphology of the apoptotic bodies, the preferred programme cell
literature which suggests that chalcones can induce apoptosis in
death [26e28].
cancer cells. [38e47]
2.2.2. Chalcones induce cell death through apoptosis rather than
necrosis in cancer cells
2.2.3. Chalcone 38 induces early apoptosis in cancer cells
To further understand the stage of apoptosis induced by the
chalcone 38 on HT-29, we subjected the cancer cells treated with
the chalcone 38 to double staining of acridine orange (AO)-propi-
dium iodide (PI) fluorescence microscopy. In viable cells and cells
undergone early apoptosis, most of the cells will have intact cell
membrane which excludes PI and only allows AO to cross the cell
membrane. In the late apoptosis and necrotic cells, the cell mem-
brane will be disrupted and allows both AO and PI to cross the
membranes and stain the cells with a predominance of PI fluores-
cence. Therefore, the double staining approach would able to
differentiate the viable cells (green intact nucleus), early apoptosis
cells (dense green areas of chromatin condensation in the nucleus),
late apoptosis cells (red-orange areas of chromatin condensation in
the nucleus) and necrosis cells (red-orange intact nucleus or frag-
mented cells).
For HT-29 cells treated with 1% DMSO and IC50 of the chalcone
38, fluorescence microscopic observation showed that majority of
the HT-29 cells were stained with green fluorescence with mini-
mum or negligible red fluorescence (Fig. 3). HT-29 cells treated
with the chalcone 38 exhibited a higher number of cells with a
dense green core, as compared to the HT-29 cells treated with 1%
DMSO. These observations suggest treatment with chalcone
induced early apoptosis. As a comparison, we treated HT-29 cells
Cancer cell death induced by chemotherapy could be activated
through apoptosis or necrosis of which apoptosis is the preferred
mode of cell death [29,30]. Chemoresistance to conventional
chemotherapy could also be due to the dysregulated apoptotic
pathways [31e33]. Reversal of chemo-resistance and thus
enhancement of apoptosis by drug or drug-like molecule could
potentiate apoptotic cell death in cancer [34e37].
We observed that the most prominent anti-proliferative effects
were exhibited by chalcones on HT-29, the most sensitive of all
cancer cells, with the mode of cell death yet to be identified.
Microscopic observation suggests apoptotic cell death. Therefore,
eight (chalcone 2, 4, 21, 23, 27, 32, 38 and 39) out of forty-six
chalcones that fulfilled the requirement of IC50 against HT-29
cells ꢃ 10
m
M and selectivity ratio ꢄ 5 (Table 3) were selected
for a cell death study by using the Cell Death Detection ELISAPLUS
Kit (Roche, Germany). The results showed that all chalcones
induced a higher enrichment factor in apoptosis as compared to
necrosis (Fig. 2). All enrichment factors of apoptosis induced by
chalcones were significantly different (p < 0.05) as compared with
cells treated with 1% DMSO. These selected chalcones induced up
to 4.16 fold (chalcone 38) of apoptosis on HT-29 as compared to
cells treated with 1% DMSO. The results support the above
microscopic observation in which chalcone 38 induced apoptosis.
These results were also in agreement with the findings from
with 5
mM of 5-fluorouracil, the conventional chemotherapy agent
for treating colorectal cancer patients. We observed that a large
Fig. 3. Fluorescence microscopic examination of HT-29 cells treated with the (A) solvent, (B) chalcone 38 (4.5 mM) and (C) 5-fluorouracil (5 mM). Viable cells (V) were stained green
with intact nucleus; early apoptotic (EAp) cells were stained green with chromatin condensation; apoptotic cells (LAp) were stained red with chromatin condensation; necrotic cells
(N) were stained red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

384
C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
The chalcone 38 upregulated various pro-apoptotic markers in
HT-29 cells as compared to HT-29 cells treated with 1% DMSO
(Fig. 4B). Caspase 8, an initiator cysteineeaspartic acid protease
(caspase), was the highest upregulated pro-apoptotic marker in
HT-29 cells treated with the chalcone 38 (11.000 fold). We also
observed up regulation of caspase 3 by 2.651folds in HT-29 cells,
which implies that caspase 8 dependent activation of caspase 3
results in amplification of apoptosis [55,56]. Also, we observed a
2.343 fold increase in the second mitochondrial derived activator
(SMAC) of caspase in HT-29 cells treated with the chalcone 38 as
compared to control. SMAC induces apoptosis as a result of the
activation of caspases [57]. Treatment with the chalcone 38 has
induced a high level of pro-apoptic bcl-2 markers such as bcl-2
homology 3-interacting domain death agonist (BID, 2.867 fold
increase) [58], bcl-2 like protein 4 (BAX, 2.363 fold increase)
[59,60], bcl-2 antagonist of cell death (BAD, 2.225 fold increase)
[61], and bcl-2 like protein 11 (BIM, 2.011 fold increase) [62] in HT-
29 cells treated with the chalcone 38 as compared to control. Up-
regulation of cyclin dependent kinase inhibitor 1 (p21) is associ-
ated with enhancement of apoptosis, probably through BAX [63].
The treatment of compound 38 increased p21 (3.961fold) when
compared to the control. The other pro-apoptotic markers which
had less than twice the increase included tumour necrosis factor
receptor superfamily member 5 (CD40), necrosis factor receptor
CD40 ligand (CD40L), superfamily member 6 (Fas), Fas ligand
(FasL), insulin-like growth factor binding protein (IGFBP)-1, IGFBP-
2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, tumour necrosis factor re-
ceptor superfamily 1A (sTNF-R1), cytochrome C, TNF-alpha, TNF-
beta, p27, p53, and high temperature required protein (HTRA)
(Fig. 4B).
We also observed that a great reduction in baculoviral inhibitor
of apoptosis proteins repeat containing protein 5 (survivin), one of
the inhibitor of apoptosis proteins (IAP). Survivin exerts its anti-
apoptotic effect via binding with baculoviral IAP repeat contain-
ing protein 4 (XIAP) and thus further increase XIAP’s inhibitory
effect against caspase 9 [64]. One study suggests that survivin de-
lays SMAC release during an apoptotic stimulus [65]. Treatment of
the chalcone 38 reduced the survivin’s level (2.06 fold, Fig. 4C) in
HT-29 cells as compared to the control. The unbounded XIAP could
thus results in a 4.176 ꢀ 1.56 fold increase of XIAP in cells treated
with chalcone 38. Just like other IAPs, baculoviral IAP repeat con-
taining protein 7 (livin) is one of the anti-apoptotic markers. Cancer
cells with high expression of livin are commonly associated with
poorer prognosis [66e69]. However the role of livin as solely an
anti-apoptotic protein is remain questionable as livin could have
both pro-apoptotic and anti-apoptotic effect, as per highlighted by
Abd-Elrahman and coworkers recently. Under a strong apoptotic
stimulus, livin was cleaved by caspases to result in a truncated
protein with paradoxical pro-apoptotic and tumour suppressive
effect [70]. Our results showed an increase in livin level after
treatment with the chalcone 38. Therefore the chalcone 38 induces
apoptosis in HT-29 cells by inhibiting survivin and inducing livin’s
pro-apoptotic effect. The up-regulation of heat shock proteins
(HSP) were associated with poorer clinical outcomes and anti-
apoptotic effect [71e73]. However, the treatment implication of
HSP is still under investigation because the elevated HSP could acts
as biological adjuvants to break tolerance to tumour antigens. We
observed an induction of 5.440 ꢀ 1.26 fold increase in HSP27 and
less than 2 fold increase in HSP60 and HSP70 for HT29 cells treated
with the chalcone 38. The other anti-apoptotic markers which had
less than 2 folds, were BCL-2, bcl-2 like protein 2 (BCLw), tumour
necrosis factor receptor superfamily 1B (sTNF-1B), insulin-like
growth factor 1 receptor (IGF-1sR), insulin-like growth factor 1
(IGF-I), insulin-like growth factor-2 (IGF-II) and baculoviral IAP
repeat containing protein 3 (cIAP) (Fig. 4C).
Fig. 4. The effect of the chalcone 38 on the expression of (A) death receptors, (B) pro-
apoptotic markers, (C) anti-apoptotic markers in HT-29 cells. Fold changes more than 2
is indicated with symbol (*).
portion of HT-29 cells had undergone late apoptosis (dense red
core) and/or necrosis (red fragmented cells or cells debris) after
treatment with 5-fluorouracil. These findings further supported the
results from the microscopic observation and the Cell Death
Detection ELISAPLUS kit, in which the chalcone 38 induces apoptosis
rather than necrosis in HT-29 cells.
2.2.4. Multiple apoptosis markers induced by chalcone 38 in cancer
cells
In order to further investigate the apoptotic mechanisms
induced by chalcone 38 in cancer cells, HT-29 cells treated with the
chalcone 38 and subjected to RaybioÒ Human Apoptosis Antibody
Array (Norcoss, GA, USA). We studied the effect of the chalcone 38
on the receptors for the TNF related apoptosis inducing ligand
(TRAILR). TRAIL-R1 (also known as death receptor 4) and TRAIL-R2
(also known as death receptor 5) were reported to be up-regulated
to sensitise cancer cells to TRAIL induced apoptosis [48]. The TRAIL-
R3 and TRAIL-R4 were decoy receptors lacking intracellular death
domain and therefore dysfunctional. Over-expression of TRAIL-R3
and TRAIL-R4 could possibly prevent TRAIL to induce apoptosis.
TRAILR-3 prevents the formation of TRAIL-R2 associated death
inducing signalling complex (DISC). On the other hand, TRAIL-R4
binds to TRAIL-R2 DISC to obstruct the recruitment of caspases
within DISC as well as activates NF-kB to inhibit TRAIL induced
apoptosis [49e52]. Over-expression of the tumour necrosis factor
receptor superfamily member 21 (DR6 or death receptor 6) also
induces apoptosis [53,54]. Our results showed that HT-29 cells
treated with the chalcone 38, showed 2.221 and 2.696 fold in-
creases of TRAIL-R1 and TRAIL-R2 respectively as compared to cells
treated with 1% DMSO (Fig. 4A). The chalcone 38 did not affect the
expression of death receptor 6 (DR6), TRAIL-R3 and TRAIL-R4.

C.W. Mai et al. / European Journal of Medicinal Chemistry 77 (2014) 378e387
385
3. Conclusions
described previously [78]. All the chalcones were tested in the
concentration range of 1e100 M.
m
A series of chalcones containing electron-withdrawing and
electron-donating substituents was synthesised as a continuation
of our ongoing anticancer development research project. The target
compounds were evaluated for antiproliferative activities against
ten human TRAIL-resistant cancer cells and their selectivity indices.
A structure activity relationship (SAR) study has been made to
correlate between the chalcones structures and antitumour activ-
ities. Chalcones 2, 4, 21, 23, 27, 32, 38 and 39 possessing sub-
stituents eNH2 on ring A and eOH and eNO2 on ring B showed
potent and selective antitumour activity.
In addition, chalcone 38, the most potent, induces apoptosis in
TRAIL-resistant human colon cancer (HT-29) cells by increasing the
death receptors expression (TRAIL-R1 and TRAIL-R2) and pro-
apoptotic markers (p21, BAD, BIM, BID, BAX, SMAC, caspase 3 and
caspase 8) as well as reducing the anti-apoptotic markers (livin,
XIAP and HSP27).
4.2.4. Detection of mode of cancer cells deaths by quantitative
sandwich enzyme immunoassay (ELISA)
The degree of mode of cancer cell deaths induced by chalcones
were quantified using the Cell Death Detection ELISAPLUS kit (Roche
Diagnostics, Indianapolis, USA) as per the manufacturer’s instruc-
tion and in our previous studies [79,80].
4.2.5. Acridine orange (AO)-propidium iodide(PI) double staining
fluorescence microscopy
In order to further understand the degree of morphological
changes, HT-29 cells were challenged with chalcone 38 for 72 h and
stained with a mixture of acridine orange (AO)-propidium iodide
(PI) as described in the literature [81e83]. The stained cells were
observed under a Nikon Eclipse 80i fluorescence microscope
(Tokyo, Japan). Viable cells are presented as a green intact nucleus;
early apoptosis cells are presented as dense green areas of chro-
matin condensation in the nucleus; late apoptosis cells exhibit
dense red-orange areas of chromatin condensation in the nucleus
while necrosis cells have a red-orange intact nucleus or fragmented
cells.
The high potency of chalcone 38 over the ten tested TRAIL-
resistant tumours, in silico studies which predicted oral bioavail-
ability, and regulation of 43 apoptosis related proteins would pave a
way for future development of anticancer agents.
4. Experimental
4.2.6. Cellular apoptotic markers analysis using antibody array
Apoptosis or programmed cell death is a complex mechanism
with multiple possible pathways involved. RaybioÒ Human
Apoptosis Antibody Array (Norcoss, GA, USA) was thus used to
simultaneously determine the effect exerted by chalcone 38 on HT-
29 cells. All reagents and chemicals were supplied by the manu-
facturer unless specified. The array was prepared in accord with the
manufacturer’s instructions. In brief, HT-29 cells were seeded for
24 h and treated with chalcone 38 at IC50 or 1% DMSO (negative
control) for 3 days. Cells were rinsed with cold phosphate buffer
saline (PBS) (SigmaeAldrich, St Louis, MO, USA) to remove any dead
cells or debris. Cells were lysed using the 1 ꢅ Lysis Buffer containing
Protease Inhibitor Cocktail as supplied by the manufacturer. Cells
were re-suspended through gentle pipetting and subsequently the
lysates were rocked gently for 30 min at 4 ꢁC. The mixture was
centrifuged at 14,000ꢅ g for 10 min. The protein lysates (super-
natant) were collected and the protein concentration was esti-
mated using a Bio-Rad Protein Assay Kit (Hercules, CA, USA). All the
4.1. Chemistry
A total of 46 chalcone derivatives (1-46, Table 1) were syn-
thesised by following a ClaiseneSchmidt condensation reaction
[74,75]. The Lipinski’s rule of five [76,77] parameters for all the
chalcones were predicted using Discovery Studio 2.5, Accelrys (San
Diego, CA, USA). The details of synthesis and spectroscopic data are
presented in the supplementary information.
4.2. Biological activity
4.2.1. Cell lines
The human oestrogen receptor positive breast cancer cells
(MCF7; HTB-22Ô), human oestrogen receptor negative breast
cancer cells (MDA-MB-231; HTB-26Ô), human cervical cancer cells
(HeLa; CCL-2Ô), human ovarian cancer cells (Caov-3; HTB-75Ô),
human lung cancer cells (A549; CCL-185Ô), human liver cancer
cells (Hep G2; HB-8065Ô), human colorectal cancer cells (HT-29;
HTB-38Ô), human erythromyeloblastoid leukaemia cells (K-562;
CCL-243Ô), and non-cancerous human embryonic kidney cells
(HEK-293; CRL-1573Ô) were obtained from the American Tissue
Culture Collection (Rockville, MD, USA); while human T-lympho-
blastoid leukaemia cells (CEM-SS) was obtained from the NIH AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH: CEM-SS (Cat# 776) from Dr. Peter L. Nara. Human nasopha-
ryngeal cancer cells (CNE-1) were a kind gift from Professor Sam C.
K. (Institute of Biological Sciences, University of Malaya, Malaysia).
lysates were diluted with the 1ꢅ Blocking Buffer to 200
mg/mL. The
diluted lysates were added to the antibody array membranes and
incubated for 2 h at room temperature. The membranes were
washed using 1ꢅ Wash Buffer I and 1ꢅ Wash Buffer II as specified
by the manufacturer. A cocktail of biotin-conjugated and anti-
apoptotic protein antibodies were incubated with the membranes
for 2 h at room temperature. Remaining unbound antibodies were
washed away by rinsing buffers before the membranes were
incubated in the dark with horseradish peroxidase (HRP)-strepta-
vidin for 2 h at room temperature. The arrays were scanned using
the Axon Gene Pix 4000B (Molecular Devices, Sunnyvale, CA, USA)
and the values were extracted using the Axon Gene Pix Pro 6.1
(Molecular Devices, Sunnyvale, CA, USA) software. The data was
then analysed using the RayBioÒ Analysis Tool (RayBiotech, Nor-
cross, VA, USA).
4.2.2. Cell culture
All cancerous and non-cancerous cells were cultured in Dul-
becco’s Modified Eagle’s Medium supplemented with 10% foetal
bovine serum, 100IU/mL of penicillin, 100 mg/mL of streptomycin,
with L-glutamine, 4.5 g/L glucose and sodium pyruvate (Sigmae
Aldrich, St Louis, MO, USA). All cells were maintained at 37 ꢁC under
5% CO2 in a humidified incubator.
4.3. Statistical analysis
All data were reported as mean ꢀ standard deviation from a
minimum three independent experiments. Statistical significant
difference was analysed using one-way analysis of variance
(ANOVA) or independent sample T-test through SPSS (version 18.0)
4.2.3. Cell proliferation assay
Inhibition of cell proliferation by chalcones was determined
using the methyl thiazolyl tetrazolium (MTT) cell viability assay, as

386
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We would like to thank the International Medical University for
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