Carbazole Aminoalcohols Induce Antiproliferation and Apoptosis of Human Tumor Cells by Inhibiting Topoisomerase I

Article abstract of DOI:10.1002/cmdc.201600391

Novel carbazole aminoalcohols were designed and synthesized as anticancer agents. Among them, alkylamine-chain-substituted compounds showed the most promising antiproliferative activity, with IC₅₀values in the single-digit micromolar range against two human tumor cell lines. Topoisomerase I (topo I) is likely to be one of the targets of these compounds. Results of comet assays and molecular docking indicate that the representative compounds may act as topo I poisons, causing single-strand DNA damage by stabilizing the topo I–DNA cleavage complex. In particular, the most potent compound, 1-(butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (6), was shown to be able to induce G₂-phase cell-cycle arrest and apoptosis in HeLa cells.

Full text of DOI:10.1002/cmdc.201600391

DOI: 10.1002/cmdc.201600391
Full Papers
Carbazole Aminoalcohols Induce Antiproliferation and
Apoptosis of Human Tumor Cells by Inhibiting
Topoisomerase I
Weisi Wang+,[a, c] Xiao Sun+,[b] Deheng Sun,[b] Shizhu Li,[a] Yang Yu,[b] Tingyuan Yang,[b]
Junmin Yao,[a] Zhuo Chen,*[b] and Liping Duan*[a]
Novel carbazole aminoalcohols were designed and synthesized
as anticancer agents. Among them, alkylamine-chain-substitut-
ed compounds showed the most promising antiproliferative
activity, with IC50 values in the single-digit micromolar range
against two human tumor cell lines. Topoisomerase I (topo I) is
likely to be one of the targets of these compounds. Results of
comet assays and molecular docking indicate that the repre-
sentative compounds may act as topo I poisons, causing
single-strand DNA damage by stabilizing the topo I–DNA cleav-
age complex. In particular, the most potent compound, 1-(bu-
tylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (6), was
shown to be able to induce G2-phase cell-cycle arrest and
apoptosis in HeLa cells.
Introduction
Eukaryotic topoisomerase I (topo I) was first discovered by
Champoux and Dulbecco in 1972.[1] Topo I is able to relax su-
percoiled DNA and plays an essential role in DNA replication
and transcription.[2,3] Topo I inhibitors are divided into two cat-
egories: topo I poisons and catalytic inhibitors. Topo I poisons
stabilize the topo I–DNA cleavage complex and lead to the ac-
cumulation of DNA damage. In terms of chemical structure,
most of topo I poisons share a common feature: a planar aro-
matic scaffold and an amine side chain (Figure 1).
tinct scaffolds is an attractive drug development strategy for
tumor treatment.[8–10]
Carbazole is a prominent core structure found in numerous
natural and synthetic compounds with a wide range of biologi-
cal activities, including antimalarial, antineoplastic, and neuro-
protective effects.[11–13] In view of anticancer agents, many car-
bazole derivatives have been shown to exhibit antitumor activ-
ity by targeting diverse enzymes or kinases.[14–16] However, few
studies have addressed the relationships between topo I and
carbazoles.[17] More generally for carbazole compounds, carba-
zole aminoalcohols have been reported as potential antimalari-
al agents[18] and neurological regulators,[19] while their antitu-
mor capacity is unknown.
Camptothecin (CPT), extracted from Camptotheca acuminate,
is a classic topo I poison, which binds to the topo I–DNA cleav-
age complex and stabilizes it, thereby inducing DNA
damage.[4,5] At present, CPT analogues irinotecan and topote-
can are first-line anticancer drugs, which are widely used for
treating small-cell lung cancer and advanced cervical cancer.[6]
However, CPT analogues have well-known limitations, including
chemical instability, dose-limiting side effects, and drug resist-
ance.[6,7] Thus, the discovery of novel topo I inhibitors with dis-
In this study, a series of carbazole aminoalcohol derivatives
were designed and synthesized. Their cytotoxicity against two
human cancer cell lines was evaluated. Additionally, because
carbazole aminoalcohols possess the common structural fea-
ture of topo I poisons, it is reasonable to hypothesize that
topo I is one of the targets of carbazole aminoalcohols. Ac-
cordingly, the topo I inhibitory activity of target compounds
was also evaluated, and structure–activity relationships (SARs)
are discussed herein. Comet assays and molecular docking
analyses were used to predict the potential mode of action of
these compounds. Furthermore, apoptosis and cell-cycle analy-
ses were performed to reveal the antitumor mechanisms of
the most potent compounds.
[a] Dr. W. Wang,+ Dr. S. Li, J. Yao, Dr. L. Duan
National Institute of Parasitic Diseases, Chinese Center for Disease Control
and Prevention, WHO Collaborating Centre for Malaria, Schistosomiasis,
and Filariasis, Key laboratory of Parasitology and Vector Biology of the Chi-
nese Ministry of Health, Shanghai 200025 (China)
E-mail: duanlp1@chinacdc.cn
[b] X. Sun,+ D. Sun, Y. Yu, T. Yang, Dr. Z. Chen
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East
China University of Science and Technology, Shanghai 200237 (China)
E-mail: chenzhuo@ecust.edu.cn
Results and Discussion
[c] Dr. W. Wang+
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceuti-
cal Sciences, Zhejiang University, Hangzhou 310058 (China)
Chemistry
[+] These authors contributed equally to this work.
The synthetic routes of carbazole aminoalcohol derivatives are
summarized in Scheme 1. Reaction of carbazole (1a–c) and
epoxy chloropropane in the presence of KOH afforded the
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600391.
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Figure 1. Structures of topo I poisons.
Topo I inhibition
Taking into account the structural similarity between carbazole
aminoalcohols and known topo I poisons, we theorized that
the former could serve as a class of topo I inhibitors. To test
this hypothesis, topo I-mediated DNA relaxation assays were
performed to examine their topo I inhibitory activity. CPT was
used as the reference compound.
Scheme 1. Synthetic routes for target compounds 3–18. Reagents and condi-
tions: a) KOH, DMF, 08C, 3 h; b) amines (RNH2 or RR’NH), BiCl3, EtOH, reflux,
6 h, 50–71% (over two steps).
In the preliminary assay (Figure 2A), compounds with alkyla-
mine tails (5–7) displayed significant topo I inhibitory activity
epoxy propane intermediates 2a–c, which subsequently react-
ed with appropriate amines to obtain the corresponding
target compounds 3–18.
In vitro cytotoxicity
All the synthesized compounds were evaluated for their cyto-
toxicity against two human cancer cell lines: HeLa (human cer-
vical carcinoma) and HL60 (human promyelocytic leukemia).
The results are summarized in Table 1. In general, all test com-
pounds exhibited moderate cytotoxicity, with IC50 values in the
micromolar range. The derivatives with aliphatic amino and
benzylamino groups (3–7, and 15–18) were slightly more
potent than those possessing aromatic amino groups (8–14) in
both cell lines. Among them, alkylamine tails (in 5–7) were
found to be the preferred substituents for cytotoxicity. n-Buty-
lamino-substituted compound 6 exhibited the best cytotoxicity
against HeLa and HL60 cells (IC50: 3.64ꢀ0.65 and 4.58ꢀ
0.54 mm, respectively). In addition, by varying the X-group sub-
stituents of the carbazole core, we found the dichlorinated car-
bazole to act as a privileged core structure. Eliminating the
chlorine atoms (in 17) or replacing them with bromine atoms
(in 18) resulted in a two- to three-fold decrease in potency, re-
spectively.
Figure 2. Inhibition of topo I-mediated DNA (pBR322) relaxation by carba-
zole aminoalcohols (see Experimental Section for details). D=pBR322,
T=pBR322+topo I, C=pBR322+topo I+CPT. A) Compounds 3–18 and
CPT were incubated with topo I and pBR322 at 100 mm. B) Compounds 5, 6,
and 7 were incubated with topo I and pBR322 at concentrations of 10, 20,
50, and 100 mm.
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at a concentration of 100 mm, which were more effective than
CPT. In addition, compounds 3, 4, and 18 exhibited high inhibi-
tory activity as evidenced by their IC50 values in the low micro-
molar range. These results are in agreement with the tendency
of antiproliferative activity. In contrast, the derivatives with aro-
matic amino substituents (8–14) did not achieve satisfactory
performance in topo I inhibition studies, and this result is also
in accordance with their relatively weak cytotoxicity. It seems
that steric effects and the basicity of the nitrogen atom of the
amine tail affect potency, especially for topo I inhibitory activi-
ty. The use of small or flexible groups rather than bulky sub-
stituents as amino tails was beneficial for activity. Introducing
more basic amino tails also had a positive effect on potency.
Further investigation revealed that compounds 5–7 inhibit
topo I in a dose-dependent manner (Figure 2B). In particular,
at a concentration of 50 mm, all three compounds exhibited
more potent topo I inhibitory ability than CPT (100 mm).
Table 1. In vitro cytotoxicity of carbazole aminoalcohols 3–18.
Compd
X
R
IC50 [mm][a]
HeLa
HL60
3
4
Cl
Cl
8.73ꢀ2.92
9.16ꢀ1.69
7.54ꢀ1.03 10.22ꢀ3.39
5
6
7
Cl
Cl
Cl
9.13ꢀ3.91
3.64ꢀ0.65
6.43ꢀ1.61
8.87ꢀ3.48
4.58ꢀ0.54
5.48ꢀ2.09
DNA damage in HeLa cells
8
9
Cl
Cl
27.20ꢀ7.48 15.00ꢀ7.91
25.37ꢀ4.78 16.11ꢀ1.76
Inhibition of topo I, especially drug trapping of the topo I–DNA
cleavage complex (like topo I poisons), frequently results in
DNA damage, which triggers cell death. We therefore used
comet assays to examine whether compounds 5–7 could
induce an accumulation of DNA damage in HeLa cells. Accord-
ing to the results (Figure 3A), relative to negative control sam-
ples, a significant increase in DNA damage was observed in
HeLa cells after treatment with 5–7 and CPT at a concentration
of 10 mm. Tail DNA reveals the actual DNA damage. The greater
percentage of DNA in the tail implies more DNA damage.[20]
Notably, the olive tail moments of compounds 6 and 7 were
34.9 and 39.8%, respectively, about two-fold more than the
extent induced by CPT (18.7%, Figure 3B). The results indicat-
ed that, in HeLa cells, compounds 6 and 7 cause DNA damage
more efficiently than CPT under the same conditions.
10
Cl
15.58ꢀ4.04 13.58ꢀ3.70
11
12
13
Cl
Cl
Cl
25.86ꢀ1.59 19.51ꢀ1.15
27.76ꢀ3.65 16.92ꢀ3.01
32.21ꢀ4.97 13.60ꢀ3.34
14
15
Cl
Cl
22.03ꢀ4.21 14.64ꢀ6.20
10.41ꢀ1.54 10.04ꢀ2.55
16
17
Cl
H
8.27ꢀ1.01
7.15ꢀ3.12
10.30ꢀ1.43 11.10ꢀ4.49
9.84ꢀ4.94 10.08ꢀ3.48
18
Br
–
CPT
–
NT[b]
0.020ꢀ0.003
Figure 3. Comet assays in HeLa cells and olive tail moment analyses.
A) Comet assays in HeLa cells in the presence of compounds 5–7 and CPT at
10 mm. B) Olive tail moment of each compound. Ten comets chosen random-
ly from six different pictures were calculated by software and treated statisti-
cally; ***p<0.001 relative to untreated control.
[a] Data are then meanꢀSD of three independent experiments. [b] Not
tested.
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Molecular docking studies
Table 2. Cell-cycle distribution of HeLa cells after treatment with com-
pound 6.
In an attempt to understand the molecular basis for topo I in-
hibition, molecular docking analyses were performed to pre-
dict the binding modes of representative compound 6 (R and
S isomers) with topo I by using the C-DOCKER program within
the Discovery Studio 2.1 software package. The published X-
ray crystal structure of the topo I–DNA–topotecan complex
(PDB ID: 1K4T) was used for docking calculations.
Sample
Phase
G1 [%]
Sub-G1 [%]
S [%]
G2 [%]
Control
5.34
8.87
6.37
12.93
49.41
45.27
42.38
49.67
47.16
29.16
29.01
28.63
23.84
17.68
13.24
12.50
13.90
16.06
18.33
8.25
6 (0.5 mm)
6 (1 mm)
6 (5 mm)
6 (10 mm)
Similar binding modes of (R)- and (S)-6 with the topo I–DNA
cleavage complex were obtained in the docking studies
(Figure 4). Like CPT and topotecan, (R)- and (S)-6 were also
As summarized in Table 2, a dose-dependent increase in G2
and sub-G1 populations was observed in response to the treat-
ment by compound 6. G2-phase arrest usually occurs in re-
sponse to DNA damage, preventing the transmission of
damage to daughter cells.[21] The percentage of cells in G2 and
sub-G1 phases increased remarkably after treatment with com-
pound 6 at 5 mm, from 12.5 to 18.3% and 5.3 to 12.9%, respec-
tively. At a concentration of 10 mm compound 6, around half
of the cells entered the sub-G1 phase (apoptosis). The results
indicate that 6 induces G2-phase cell-cycle arrest followed by
apoptosis in HeLa cells.
Consistent with the dramatically increased sub-G1 cell popu-
lation occurring in cell-cycle analysis, a dose-dependent in-
crease in the percentage of apoptotic and dead cells was also
observed in the apoptosis assay (Figure 5). Annexin V-FITC
analysis revealed a basal apoptotic population of 6.1% in the
untreated culture. After incubation for 24 h, compound 6
(20 mm) induced almost all of the HeLa cells into the apoptotic
stage (43.9% in late stage and 55.2% in early stage). Thus,
apoptosis is the primary mode of cell death induced by 6.
Figure 4. Molecular docking analysis of (R)- and (S)-6 with the topo I–DNA
cleavage complex. A) 3D schematic interaction model of (R)-6 (magenta) and
the topo I–DNA complex; B) binding mode of (R)-6 with contacting residues
and base pairs of the topo I–DNA complex; C) 3D schematic interaction
model of (S)-6 (green) and the topo I–DNA complex; D) binding mode of (S)-
6 with contacting residues and base pairs of the topo I–DNA complex. Hy-
drogen bonds are highlighted as green dashes.
found to intercalate into DNA at the DNA cleavage site and to
form base-stacking interactions with downstream (ꢁ1) T:A and
upstream (+1) G:C base pairs (Figure 4A,C). In addition, both
isomers formed one hydrogen bond with Asp533, a residue
near the active site, which is known to be necessary for topo I
sensitivity to CPT.[4] The hydrogen bond occurs between the ni-
trogen atom of the amine tail of 6 (donor) and carboxylate ion
of Asp533 (acceptor, Figure 4B,D). The predicted binding
modes are consistent with our experimental data, indicating
that compound 6 stabilizes the topo I–DNA cleavage complex
like CPT, and probably acts as a topo I poison.
Induction of cell-cycle arrest and apoptosis
As illustrated above, the representative derivatives 5–7 could
effectively inhibit topo I, induce DNA damage, and inhibit the
proliferation of multiple cancer cell lines. Thus, the most
potent compound 6 was selected for further studies of its
effect on cell-cycle progression and the induction of apoptosis
in HeLa cells.
Figure 5. Apoptotic cells were detected by Annexin V/PI double staining
after incubation with compound 6 (5, 10, and 20 mm) for 24 h.
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extracted with EtOAc (3ꢂ10 mL). The organic phase was washed
with water (3ꢂ20 mL) and brine (3ꢂ20 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The obtained
residues were purified by recrystallization from ethanol to afford
target compounds 3–18. The structural characterization of target
compounds is described in the Supporting Information.
Conclusions
In this study, carbazole aminoalcohol derivatives were synthe-
sized and evaluated as a series of novel antitumor agents. SAR
studies revealed that the aliphatic-amine-substituted deriva-
tives are more potent antitumor agents than those with aro-
matic amino groups. Among them, alkylamine-substituted
compounds 5–7 exhibited the most efficient antiproliferative
activity, which is in accordance with their topo I inhibitory abili-
ty. Through comet assays and molecular docking analyses, we
speculate that the representative compounds 5–7 act as topo I
poisons, which cause single-strand DNA damage by stabilizing
the topo I–DNA cleavage complex. Furthermore, the most
potent compound 6 induced G2-phase arrest and apoptosis in
HeLa cells. This study identified the antitumor activity of
a series of carbazole aminoalcohols and discovered a novel
topo I inhibitor scaffold, which may be promising for the de-
velopment of new anticancer agents.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(piperidin-1-yl)propan-2-ol
(3): white solid (66%, over two steps), mp: 140.3–141.38C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(2-methylpiperidin-1-yl)pro-
pan-2-ol (4): white solid (70%, over two steps), mp: 131.8–
133.48C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(propylamino)propan-2-ol (5):
white solid (71%, over two steps), mp: 117.6–120.18C.
1-(Butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (6):
white solid (50%, over two steps), mp: 116.9–118.18C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(pentylamino)propan-2-ol (7):
white solid (59%, over two steps), mp: 98.5–99.68C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(phenylamino)propan-2-ol
(8): white solid (59%, over two steps), mp: 140.3–142.08C.
Experimental Section
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-((4-fluorophenyl)amino)pro-
pan-2-ol (9): white solid (53%, over two steps), mp: 137.7–
140.38C.
Chemistry
Reagents and solvents were purchased from Sigma–Aldrich and
were used without further purification. Melting points were deter-
mined with a B-540 Bꢁchi apparatus and are uncorrected. H and
1-((4-Bromophenyl)amino)-3-(3,6-dichloro-9H-carbazol-9-yl)pro-
pan-2-ol (10): white solid (43%, over two steps), mp: 154.8–
157.48C.
1
13C NMR spectra were recorded on a Bruker AM-400 spectrometer
(400 MHz). Chemical shifts (d) are given in ppm relative to TMS as
internal standard, and signals are denoted with the following ab-
breviations: s, singlet; d, doublet; t, triplet; m, multiplet. Mass
spectra (MS, ESI) were recorded on a Thermo Q Exactive Orbitrap
LC–MS/MS instrument. Thin-layer chromatography (TLC) was car-
ried out using plate silica gel F254 (Merck). All yields are unopti-
mized and generally represent the result of a single experiment. El-
emental analyses (C, H, and N) were undertaken using an Elemen-
tar Vario ELIII analyzer. Analytical HPLC was performed on a Waters
2695-2996 HPLC system and an Elite hypersil BDS C18 column
(5 mm, 4.6ꢂ250 mm) using the following binary solvent system:
CH3CN/0.1% aqueous trifluoroacetic acid (TFA)=85:15; flow rate:
1.0 mLminꢁ1, l=254 nm, column temperature=308C. The purities
of all test compounds determined by HPLC were >95%.
1-((4-Chlorophenyl)amino)-3-(3,6-dichloro-9H-carbazol-9-yl)pro-
pan-2-ol (11): white solid (71%, over two steps), mp: 139.6–
143.08C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol
(12): white solid (66%, over two steps), mp: 157.9–160.58C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-(o-tolylamino)propan-2-ol
(13): white solid (69%, over two steps), mp: 108.7–111.68C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-((4-methoxyphenyl)amino)-
propan-2-ol (14): white solid (63%, over two steps), mp: 164.6–
167.48C.
1-(Benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol
(15): white solid (68%, over two steps), mp: 139.8–142.58C.
1-(3,6-Dichloro-9H-carbazol-9-yl)-3-((3,4-dimethoxyphenethyl)a-
mino)propan-2-ol (16): white solid (61%, over two steps), mp:
138.5–139.48C.
General procedures for the synthesis of 9-(oxiran-2-ylmethyl)-
9H-carbazoles (2a–c): To a cooled solution of compounds 1a–
c (2 mmol) in DMF (20 mL, 08C), KOH (135 mg, 2.4 mmol) was
added slowly. After stirring for an additional 1 h, epoxy chloropro-
pane (2.4 mmol) was added dropwise. The progress of the reaction
was monitored by TLC. The reaction was quenched with water
(30 mL). The resulting mixture was extracted with ethyl acetate (3ꢂ
20 mL). Organic layer was washed with water (3ꢂ50 mL) and brine
(3ꢂ50 mL), dried over anhydrous Na2SO4. The solvent was evapo-
rated under reduced pressure and dried under high vacuum over-
night. The solid was used directly in the epoxide opening reaction
without further purification.
1-(Butylamino)-3-(9H-carbazol-9-yl)propan-2-ol (17): white solid
(58%, over two steps), mp: 114.2–115.18C.
1-(Butylamino)-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-ol (18):
white solid (53%, over two steps), mp: 123.4–124.88C.
Biological evaluation
General procedures for the synthesis of carbazole aminoalco-
hols (3–18): To a solution of 9-(oxiran-2-ylmethyl)-9H-carbazole
(2a–c, 2 mmol) in EtOH (20 mL), corresponding amines (6 mmol)
was added. For low reactive amines (e.g., arylamines), BiCl3
(1 mmol) was also added. The reaction mixture was heated at
reflux for 6 h. The progress of the reaction was monitored by TLC.
After reaction, the mixture was quenched with water (10 mL) and
Cell culture: HeLa and HL60 cell lines were obtained from Shang-
hai Institute of Meteria Medica, Chinese Academy of Sciences.
HeLa cells were cultured in adherence in MEM (Gibco, USA) con-
taining 10% fetal bovine serum (Lanzhou Lark, China), 1% sodium
pyruvate (Sigma, USA) and 1% GlutaMAX-I (Gibco, USA). HL60 cells
were cultivated in suspension in RPMI-1640 (HyClone, USA) con-
taining 10% fetal bovine serum. Cultures were performed in a hu-
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midified incubator (Thermo, USA) in an atmosphere of 5% CO2 at
378C.
ase I–DNA–topotecan complex (PDB ID: 1K4T) was used for the
docking calculation. After removing the ligand and solvent mole-
cules, the CHARMm force field was applied to the protein, and the
area around intercalation sites was chosen as the active site with
a radius set as 9 ꢃ. At physiologically relevant pH (7.4), both nitro-
gen atoms of 6 are protonated (see Figure S1 in the Supporting In-
formation). Accordingly, they were charged at +1 for docking sim-
ulations. Each isomer of 6 was generated random conformations
using CHARMm-based molecular dynamics (1000 steps), and then
docked into the defined binding site. The other parameters were
set as default. The final binding conformations of (R)- and (S)-6
were determined based on the calculated CDOCKING energy and
visual check. The most stable binding mode among the top 10
docking poses of each isomer is presented in Figure 4.
Cytotoxicity assay: Sulforhodamine B (SRB) and MTT assays were
performed on adherent and suspended cells, respectively. HeLa
and HL60 cells were planted into 96-well plates at a densities of
3000 and 6000 cells per well, respectively. After incubation for
24 h, cells were treated with compounds at gradient concentra-
tions for 72 h. For the SRB assay, cells were fixed with 10% tri-
chloroacetic acid, and then stained with SRB (TCI, Japan). Free dye
was washed by 1% acetic acid solution. Conjunct SRB was dis-
solved by 10 mm Tris solution. Plates were measured at l=560 nm
on a Synergy 2 Multi-Mode Reader (BioTek, USA). For MTT assays,
20 mL of 5 mgmLꢁ1 MTT (Amresco, USA) solution was added to
each well and incubated for 4 h to form formazan. Formazan was
incubated by Lysis solution (10% SDS, 5% isobutyl alcohol,
0.012 mm HCl) overnight. Plates were examined at l=570 nm. The
inhibition rate was calculated by the following formula [Eq. (1)]:
Cell-cycle analysis by flow cytometry: Cell-cycle analysis was per-
formed with PI/RNase solution purchased from Sungene. HeLa
cells were planted into a six-well plate at a density of 2ꢂ105 cells
per well. Cells were treated with compounds for 24 h and then har-
vested. After washing with D-Hanks solution twice, cells were fixed
with 70% ethyl alcohol at 48C overnight. The next day, samples
were washed and stained with PI/RNase solution for 30 min at
room temperature. Samples were then analyzed on a flow cytome-
ter (BD Biosciences, USA). And data were treated with FlowJo.
ꢀ
ꢁ
ODtreated ꢁ ODblank
ODcontrol ꢁ ODblank
Inhibition rate ¼ 1 ꢁ
ꢂ 100 %
ð1Þ
IC50 values were then calculated.
Topo I-mediated DNA relaxation assay: The topo I inhibitory
action of compounds was tested by relaxing supercoiled pBR322
DNA. DNA topoisomerase I assay kit and pBR322 DNA were pur-
chased from TAKARA. 0.05 mg pBR322 DNA, 0.05 U topoisomerase I,
2 mL 0.1% BSA, and indicated concentrations of compounds were
added in diluted assay buffer to a volume of 20 mL, and incubated
for 30 min at 378C. The reaction was started by adding topoiso-
merase I, and stopped by 2 mL 10% SDS and 2 mL DNA loading
buffer (TianGen, China). Samples were electrophoresed in 1% agar-
ose gel in TAE buffer for 2 h at 50 V. Gel was stained with
5 mgmLꢁ1 ethidium bromide for 10 min, and photographed at
254 nm UV.
Apoptosis analysis by flow cytometry: Annexin V-FITC Cell Apop-
tosis Analysis Kit was purchased from Sungene. After the same
treatment on HeLa cells (see cell-cycle analysis), cells were collect-
ed and washed three times with D-Hanks and then washed with
binding buffer. Samples were stained with Annexin V-FITC solution
and PI solution. Samples were analyzed by flow cytometry.
Statistical analysis: Data are expressed as the meanꢀSD unless
otherwise indicated. Statistical analysis of data for multiple groups
was performed with one-way analysis of variance (ANOVA). Stu-
dent’s t-test was applied for the comparison of two groups, and
p values <0.01 are considered significant.
Comet assay: Single-strand DNA damage was evaluated by comet
assay. HeLa cells were seeded in a six-well plate at a density of 2ꢂ
105 cells per well. After incubation for 24 h, cells were treated with
indicated concentrations of compounds for 24 h, and collected
and washed three times with D-Hanks solution. The cell density
was adjusted to 105 cells per mL; 5 mL of cells were mixed with
75 mL of 0.5% low-melting-point agarose (Amresco, USA) which
was prepared and sub-packaged previously and was melted and
kept warm at 378C before the experiment. The mixture was spread
on a 1% NMA coated slide with a coverslip and solidified on ice.
The coverslip was removed and the slide was immersed into cold
fresh lysis solution (2.5m NaCl, 100 mm EDTA, 10 mm Tris, 1%
Triton X-100, 10% DMSO) for 2 h at 48C in the dark. The slide was
removed from the lysis solution and washed in Tris solution for
5 min three times, and then unwinding DNA in electrophoresis
buffer (300 mm NaOH, 1 mm EDTA, pH>13, made freshly) for
30 min at 48C in the dark. The slide was electrophoresed for
25 min at 15 V at 48C in the dark. The slide was neutralized with
Tris·HCl buffer (0.4m Tris, pH 7.5) for 5 min three times, and dried
with anhydrous ethyl alcohol. The slide was stained with SYBR
Green I (Life, USA) before observation, and photographed under
blue light with a Ti-S Fluorescence microscope (Nikon, Japan).
Olive tail moment was statistically treated with CASP (Comet As-
say Software Project).
Acknowledgements
This work was supported financially by the Fundamental Re-
search Funds for the Central Universities (China), the National
Natural Science Foundation of China (Grant Nos. 21302054 and
21502181; Z.C. and W.W.), the Shanghai Committee of Science
and Technology (Grant No. 13ZR1453100; Z.C.), and the Opening
Fund of Shanghai Key Laboratory of Chemical Biology (Grant No.
SKLCB-2012-05; Z.C.).
Keywords: antitumor agents
aminoalcohols · cell-cycle arrest · topoisomerase I
·
apoptosis
·
carbazole
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Manuscript received: August 3, 2016
Revised: October 24, 2016
Final Article published: && &&, 0000
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FULL PAPERS
W. Wang, X. Sun, D. Sun, S. Li, Y. Yu,
T. Yang, J. Yao, Z. Chen,* L. Duan*
Under arrest: Carbazole aminoalcohols
were synthesized and tested for their in-
hibitory potential against topoisomer-
ase I (topo 1) and for their antiprolifera-
tive activity against human tumor cell
lines. Structure–activity relationships in-
dicated that alkylamine-substituted
compounds exhibit the most efficient
antiproliferative activity, in agreement
with their topo I inhibitory capacity. Fur-
ther studies confirmed that the most
potent compound induces G2-phase
cell-cycle arrest and apoptosis in HeLa
cells.
&& – &&
Carbazole Aminoalcohols Induce
Antiproliferation and Apoptosis of
Human Tumor Cells by Inhibiting
Topoisomerase I
&
ChemMedChem 2016, 11, 1 – 8
www.chemmedchem.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!