Different cytotoxicities and cellular localizations of novel quindoline derivatives with or without boronic acid modifications in cancer cells

Article abstract of DOI:10.1039/c3cc45203d

The synthesis of a 4 × 4 series of novel quindoline derivatives with or without boronic acid modifications and their cytotoxicities, cellular localizations, and implications on cancer cells are presented and discussed. The Royal Society of Chemistry 2013.

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Different Cytotoxicities and Cellular Localizations of Novel Quindoline
Derivatives With or Without Boronic Acid Modifications in Cancer
Cells
Ruijuan Yin†, Meng Zhang†, Cui Hao†, Wei Wang, Peiju Qiu, Shengbiao Wan, Lijuan Zhang*, Tao
Jiang*
5
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
40 Scheme 1. Reagents, conditions and yields: (a) RNH(CH2)nNH2,
The synthesis of 4x4 series of novel quindoline derivatives
with or without boronic acid modifications and their
10 cytotoxicities, cellular localizations, and implications on
cancer cells are presented and discussed.
CH3CH2OCH2CH2OH, reflux, 17h; (b) benzoic acid
,
DMTꢀMM,
CH3CH2OCH2CH2OH, r.t., 17h; (c) 4ꢀCarboxyphenylboronic acid or 3ꢀ
Carboxyphenylboronic acid, DMTꢀMM, CH3CH2OCH2CH2OH, r.t., 17h;
45 Since cell surfaces are covered by a variety of carbohydrates, we
hypothesized that introducing boronic acid modification to
quindoline derivatives might reduce the number of the derivatives
entering the cell due to increased interactions between boronic
acid and cell surface carbohydrates based on boronolectin
50 studies[1] . As fewer boronic acid modified quindoline derivatives
get inside the cells, less cell killing should be observed. Thus, we
used two human colon cancer cell lines (HT29 and HCT116) and
two human lung cancer cell lines (H1299 and A549) to test the
hypothesis.
Chemically synthesized boronic acid derivatives[1] have been
developed into drugs[2] or biomarker identification reagents[3] that
either work inside of cells or at cell surface due to unique
15 carbohydrate interactions[4] and Lewis acidity[2] of the boronic
acid moiety at physiological conditions. However, it is unknown
if boronic acid modification has any impact on the cellular
localizations and biological activities of its parental compounds.
Quindoline has intrinsic fluorescence, penetrates cells, interacts
20 with DNAs, and kills cancer cells by three established
mechanisms[5]. Thus, we designed and synthesized 4x4 series of
novel quindoline derivatives without (2aꢀ2d and 3aꢀ3d) or with
boronic acid modifications (4aꢀ4d and 4e-4h) as shown in
Scheme 1. These derivatives were then used to evaluate the
25 impact of boronic acid modifications on the cellular localizations
and cancer cell killing properties by taking advantage of the
unique physical and biological activities of the quindoline.
To make the quindoline derivatives, the key intermediate of 11ꢀ
chloroquindoline (1) was prepared following the procedure
30 reported by Bierer[6]. The substitution reaction of compound (1)
with the commercial aliphatic diamines produced the quindoline
derivatives containing 4 different types of alkylamine sideꢀchains
at the Cꢀ11 position (2a-2d). The benzoic acid (3aꢀ3d) as well as
boronic acid modified benzoic acid derivatives (4aꢀ4d and 4e-4h)
35 were obtained by amidation of 11ꢀanimoꢀ10Hꢀindolo[3,2ꢀ
b]quinoline (2a-2d) with benzoic acid or 4ꢀcarboxyphenylboronic
acid, or 3ꢀcarboxyphenylboronic acid, respectively. The reagents,
conditions, and yield are shown in Scheme 1.
55 We first measured the percentage of viable cancer cells after 48 h
exposure to the 16 quindoline derivatives (2aꢀ2d, 3aꢀ3d, 4aꢀ4d,
and 4e-4h) at a concentration of 10 ꢁM as compared to the
compoundꢀfree control (100% viability) (Fig. 1). The results
supported our hypothesis in that all boronic acid modified
60 quindoline derivatives (4aꢀ4d and 4e-4h) were less toxic to
cancer cells than the boronic acidꢀfree quindoline derivatives (2aꢀ
2d and 3a-3d) in all cancer cell lines tested.
150
100
50
H1299
A549
1
A
2
3
4
0
a
b
c
d
a
b
c
d
B
HT29
HCT116
1
30
15
0
2
3
4
a
b
c
d
a
b
c
d
Fig.1. Percentage of viable cancer cells after 48h exposure to the quindoline
65 derivatives 2aꢀ2d, 3aꢀ3d, 4a-4d and 4e-4h at a concentration of 10 ꢁM
compared to the compoundꢀfree control (100% viability). The derivatives were
formulated initially in DMSO and then diluted in complete growth media
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(Details in Experimental Protocols) (□, 2aꢀ2d; ■, 3aꢀ3d; , 4aꢀ4d; ■, 4eꢀ4h).
(A) Effect on lung cancer cells, H1299 and A549. (B) Effect on colon cancer
cells, HT29 and HCT116
40 quindoline derivatives associated with the cancer cells.
To test this idea, we incubated the same numbers of colon cancer
cells (HT29) and lung cancer cells (A549) without (quindoline
derivativeꢀfree) or with the same amount of the derivatives 2a, 4a
and 4e (10 ꢁM) at identical cell culture conditions (24 h at 37 ºC).
45 The cells were then washed three times with PBS followed by
flow cytometry analysis. As shown in Fig. 2A, the quindoline
derivativeꢀfree cancer cells indeed showed low autoꢀfluorescent
background (HT29:12.58 ± 0.82 au vs. A549:7.02±2.93 au). In
both HT29 and A549 cells, the fluorescent intensity was
50 decreased in the order of the derivatives: 2a (HT29: 45.82 ± 5.91
au vs. A549: 30.11±7.63 au) > 4a (HT29: 23.695±1.85 au vs.
A549: 11.27±2.68au) > 4e (HT29: 21.195±1.69au vs. A549:
8.995±4.79au) > the quindoline derivativeꢀfree control (HT29:
12.58 ± 0.82 au vs. A549:7.02±2.93 au). After subtracting the
55 autoꢀfluorescent background, the fluorescent intensities in 2a, 4a
and 4e treated HT29 cells were consistently higher than that in 2a,
4a and 4e treated A549 cells. The higher fluorescent intensities
correlated perfectly with the higher cytotoxicities (Fig. 1 and
Table 1). These results indicate that the amount of the derivatives
60 associated with the cancer cells was the key to explain the
cytotoxicities of the quindoline derivatives.
5
To confirm this observation, each compound was tested in a
series of concentrations to calculate the IC50 values in each of the
four cell lines (Table 1). Interestingly, all aliphatic diamineꢀ
modified (2aꢀ2d) plus benzoic acid modified (3aꢀ3d) quindoline
derivatives showed almost the same cytotoxicities in each cell
10 line tested, indicating that different aliphatic chain length (2a, 2b,
and 2d) and structure (2c) plus benzoic acid modifications to
quindoline had almost no impact towards observed cytotoxicities.
In contrast, introducing a single boronic acid at either 3 (4e-4h)
or 4 (4aꢀ4d) position of the benzoic acid of the derivatives 3a-3d
15 resulted in greatly reduced cytotoxicities reflecting 3 to 10ꢀfold
higher IC50 values (4aꢀ4d and 4e-4h) in all four cancer cell lines
tested. Furthermore, the quindoline derivatives with 4ꢀboronic
acid modification (4aꢀ4d) had lower IC50 values than that of the
derivatives with 3ꢀboronic acid modification (4eꢀ4h), suggesting
20 that the location of boronic acid in the quindoline derivatives
played a role in the observed cytotoxicities as well (Fig. 1 and
Table 1).
Table 1. IC50 of quindoline derivatives (2a-2d, 3aꢀ3d, 4a-4h) in cancer cells
25 HT29, HCT116, A549 and H299 after 48 h exposure to the derivatives.
IC50 (ꢁm)a.
Comp.
2a
2b
2c
HT29
HCT116
0.6±0.14
0.3±0.09
0.3±0.11
0.9±0.36
0.8±0.32
1.0±0.32
0.9±0.17
1.2±0.38
2.6±0.56
1.3±0.18
2.1±0.31
1.2±0.30
3.9±0.30
2.0±0.85
15.7±0.75
1.3±0.74
A549
H1299
1.3±0.66
1.0±0.19
0.62±0.24
0.90±0.06
0.8±0.30
2.1±0.08
2.8±0.15
2.9±0.12
14.1±3.08
6.6±2.26
6.8±1.16
4.2±1.44
12.2±3.56
11.0±3.62
17.1±3.45
3.4±1.26
0.7±0.14
0.3±0.07
0.4±0.15
1.0±0.40
0.3±0.06
1.3±0.23
0.8±0.24
0.9±0.31
3.7±0.83
2.0±0.01
3.3±0.25
1.6±0.53
5.1±0.36
2.5±0.82
11.9± 4.0
1.7±0.58
2.1±0.26
1.6±0.89
1.0±0.16
2.1±0.55
1.3±0.24
2.3±0.4
2d
3a
3b
3c
2.4±0.23
3.5±0.48
17.2±2.05
7.8±1.33
14.2±1.18
16.7±6.63
29.1±1.59
14.8±1.49
23.8±3.43
18.6±5.44
3d
4a
4b
4c
4d
4e
4f
4g
4h
a. Each IC50 value was calculated from 3 independent experiments performed
in triplicate.
Fig.2. The amount of the derivatives 2a, 4a and 4e associated with the cancer
cells. HT29 (A) or A549 (B) cells were incubated with or without the
65 derivatives 2a, 4a or 4e at a concentration of 10 ꢁM. After incubation for 24h
at 37ºC, the cells were harvested, washed and then analyzed by flow cytometry.
Ten thousand events were counted for each sample. The result shown is a
representative of three independent experiments. Control: cells cultured in
complete growth medium; 2: 2a treated cells; 3: 4a treated cells; 4: 4e treated
70 cells.
Data in Fig. 1 and Table 1 showed that all quindoline derivatives
30 (2aꢀ2d, 3aꢀ3d, 4aꢀ4d, and 4e-4h) were more toxic to the two
colon cancer cell lines (HT29 and HCT116) than to the two lung
cancer cell lines (A549 and H1299), suggesting that more
quindoline derivatives might be associated with colon cancer
cells than with lung cancer cells due to different genetic
35 background, such as Pꢀglycoprotein expressing levels[7]. To test
this idea, we employed flow cytometry analysis. The logic of this
assay is that cancer cells do not have any fluorescence; only cellꢀ
associated quindoline derivatives produce fluorescence and the
intensity of fluorescence might be in proportion to the amount of
The results in Fig. 2 showed that the derivatives 2a, 4a and 4e
were associated with HT29 and A549 cells. However, flow
cytometry analysis could not precisely determine the location of
75 the derivatives associated with the cancer cells. Therefore, we
used confocal imaging analysis to further explore the issue. To
2
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40 derivatives, which has important implications in research and
development of boronic acid modified drugs.
this end, HT29 and A549 cells were incubated with the
derivatives 2a, 3a, 4a and 4e for 24 h at 37 ºC. The cancer cells
were then washed three times before confocal imaging. As shown
in Fig. 3A, high levels of green fluorescence were detected in the
2a and 3a treated HT29 cells, and the fluorescence was mostly
found around or in the cell nucleus. In contrast, the 4a and 4e
treated HT29 cells exhibited less fluorescent intensities than that
in the 2a and 3a treated HT29 cells, and the observed
fluorescence was closer to the cell membranes (Fig. 3A).
10 We also investigated the localization of the derivatives 2a, 3a, 4a
and 4e in A549 cells. The data in Fig. 3B showed that the
derivative 2a and 3a was mostly found around or in the cell
nucleus of the A549 cells, but the nuclear localization of 2a and
3a in the A549 cells was less than that in the HT29 cells (Fig.
15 3A), which was consistent with the reduced cytotoxicities and
cell association of 2a and 3a in the A549 cells than in the HT29
cells (Fig. 1, Table 1, and Fig. 2). In agreement with the
cytotoxicities and cell association data shown in Fig. 1, Table 1
and Fig. 2, the fluorescence observed in the 4a and 4e treated
20 A549 cells was less than that in the 2a and 3a treated A549 cells,
which was consistent with the decreased cytotoxicities in the
order of 2a, 3a, 4a and 4e shown in Fig. 1, Table 1, and Fig. 2.
Again, the observed fluorescence of the 4a and 4e in the A549
cell was closer to the membrane (Fig. 3B).
Notes and references
5
†
These authors contributed equally to this study.
*
School of Medicine and Pharmacy, Ocean University of China, 5
45 Yushan Road, Qingdao, China. Fax: 0086ꢀ532ꢀ82033054;
Eꢀmails: jiangtao@ouc.edu.cn and lijuanzhang@ouc.edu.cn.
† Electronic Supplementary Information (ESI) available: Experimental
procedure, compound characterization, copies of NMR. See DOI:
10.1039/b000000x/
50 ‡ This work was supported by Natural Science Foundation of China
(Grant No. 21171154 and 91129706) and Special Fund for Marine
Scientific Research in the Public Interest (01005024).
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Fig. 3. Distribution of the derivatives 2a, 3a, 4a and 4e in HT29 and A549
cells. Cells were incubated with or without the derivatives 2a, 3a, 4a or 4e at a
concentration of 10 ꢁM for 24h at 37ºC. After which, the cells were washed
three times with PBS before confocal imaging. (A) HT29 cells, (B) A549 cells.
30 Scale bar represents 10 ꢁm. Control: nonꢀderivative treated cells.
95
Conclusions
In summary, boronic acid modifications reduced quindoline’s
cytotoxicities mainly through decreased cell penetration and
nuclear localization that we attributed to the increased cell surface
35 carbohydrate interactions and/or increased difficulties in
penetrating cell membranes due to the presence of boronic acid
modification in the quindoline derivatives. This study provides
useful information about the influence of boronic acid on the
cytotoxicities and cellular localizations of the quindoline
100
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