Novel anthraquinone compounds inhibit colon cancer cell proliferation via the reactive oxygen species/JNK pathway

Article abstract of DOI:10.3390/molecules25071672

A series of amide anthraquinone derivatives, an important component of some traditional Chinese medicines, were structurally modified and the resulting antitumor activities were evaluated. The compounds showed potent anti-proliferative activities against eight human cancer cell lines, with no noticeable cytotoxicity towards normal cells. Among the candidate compounds, 1-nitro-2-acyl anthraquinone-leucine (8a) showed the greatest inhibition of HCT116 cell activity with an IC₅₀ of 17.80 μg/mL. In addition, a correlation model was established in a three-dimensional quantitative structure-activity relationship (3D-QSAR) study using Comparative Molecular Field Analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). Moreover, compound 8a effectively killed tumor cells by reactive oxygen species (ROS)-JNK activation, causing an increase in ROS levels, JNK phosphorylation, and mitochondrial stress. Cytochrome c was then released into cytoplasm, which, in turn activated the cysteine protease pathway and ultimately induced tumor cell apoptosis, suggesting a potential use of this compound for colon cancer treatment.

Full text of DOI:10.3390/molecules25071672

molecules
Article
Novel Anthraquinone Compounds Inhibit Colon
Cancer Cell Proliferation via the Reactive Oxygen
Species/JNK Pathway
Yuying Li *, Fang Guo, Yingying Guan, Tinggui Chen , Kaiqing Ma, Liwei Zhang
Zhuanhua Wang, Qiang Su, Liheng Feng, Yaoming Liu and Yuzhi Zhou
,
Key Laboratory for Chemical Biology and Molecular Engineering of Ministry of Education, Institute of
Biotechnology, Shanxi University, Taiyuan 030006, China; 201723002004@email.sxu.edu.cn (F.G.);
yanning654@126.com (Y.G.); Chentg@sxu.edu.cn (T.C.); makaiqing@sxu.edu.cn (K.M.);
lwzhang@sxu.edu.cn (L.Z.); zhwang@sxu.edu.cn (Z.W.); suqchem1984@163.com (Q.S.); lhfeng@sxu.edu.cn (L.F.);
liuym1022@sxu.edu.cn (Y.L.); zhouyuzhi@sxu.edu.cn (Y.Z.)
* Correspondence: lyy9030@sxu.edu.cn
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 10 March 2020; Accepted: 2 April 2020; Published: 4 April 2020
Abstract: A series of amide anthraquinone derivatives, an important component of some traditional
Chinese medicines, were structurally modified and the resulting antitumor activities were evaluated.
The compounds showed potent anti-proliferative activities against eight human cancer cell lines, with
no noticeable cytotoxicity towards normal cells. Among the candidate compounds, 1-nitro-2-acyl
anthraquinone-leucine (8a) showed the greatest inhibition of HCT116 cell activity with an IC50 of
17.80 µg/mL. In addition, a correlation model was established in a three-dimensional quantitative
structure-activity relationship (3D-QSAR) study using Comparative Molecular Field Analysis (CoMFA)
and comparative molecular similarity index analysis (CoMSIA). Moreover, compound 8a effectively
killed tumor cells by reactive oxygen species (ROS)-JNK activation, causing an increase in ROS levels,
JNK phosphorylation, and mitochondrial stress. Cytochrome c was then released into cytoplasm,
which, in turn activated the cysteine protease pathway and ultimately induced tumor cell apoptosis,
suggesting a potential use of this compound for colon cancer treatment.
Keywords: anthraquinone derivatives; apoptosis; 3D-QSAR; ROS-JNK; HCT116
1. Introduction
Cancer is a major cause of death in populations all over the world. Despite being the current
treatment of choice in many cases, chemotherapy still has many disadvantages, such as strong toxic
side-effects and partial lack of targeting specificity [
compounds that act specifically on target proteins in tumor cells is a focus of current research [
1
–
3
]. Therefore, the design and synthesis of new
].
4–6
Anthraquinone derivatives with a 9,10-anthracene skeleton are a class of natural compounds frequently
used in traditional Chinese medicine. These compounds have various biological properties such
as antibacterial, analgesic, antimalarial and antitumor activity [7,8]. In recent years, anthraquinone
derivatives have attracted increasing attention due to their excellent antitumor activity. Although
anthraquinone derivatives exist in a variety of plants, the extraction process is complicated and
problematic due to their low content in plants, which limits further structural optimization and
activity screening. Structural modifications are carried out on the basis of the parent nucleus to
obtain a drug with stronger efficacy and few side-effects. Indeed, some anthraquinone derivatives
have been licensed for use as antitumor drugs in the clinic. Currently marketed compounds, such
as mitoxantrone, emodin, doxorubicin and daunorubicin (Figure 1A–D), are used clinically to treat a
variety of cancers [9,10]. One of these anthraquinone antitumor drugs, mitoxantrone (MX), has been
Molecules 2020, 25, 1672; doi:10.3390/molecules25071672
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1672
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widely used in the clinic since the 1980s, and is the only relapse-remitting treatment approved by
the U.S. Federal Drug Administration for worsening of multiple sclerosis symptoms [11]. However,
clinical studies have shown that MX causes cumulative and irreversible cardiotoxicity, as most of
the MX (>95%) binds rapidly to plasma proteins when it enters the bloodstream [12]. Once there, it
causes serious toxic side-effects such as myelosuppression and gastrointestinal reactions, which limit
its clinical application [13]. Emodin is another anthraquinone obtained from natural plants that has
attracted much attention due to its many pharmacological properties [8,14]. However, due to its poor
water solubility, its use is limited and further evaluation is required to determine the full spectrum of
its pharmacological activity and toxicity.
Figure 1. Mitoxantrone (A), emodin (B), doxorubicin (C) and daunorubicin (D) that have been
used for clinical tumor treatment. The red part in the figure is the ternary coplanar structure of the
anthraquinone core.
The possibility of combining amino acids with various anti-tumor drugs to improve their water
solubility and tumor selectivity is currently under evaluation. The demand for protein-forming
amino acids in tumor cells is much higher than that of normal cells, which makes amino acids
excellent vectors for selective targeting of tumors [14]. Hsin et al. designed and synthesized a series
of 1,4-bis(2-aminoethylamino)anthraquinone-amino acid derivatives and compared their antitumor
properties to MX-amino acid derivatives [15]. Methionine anthraquinone derivatives were found to
have lower activity compared to MX-amino acids derivatives, while lysine anthraquinone derivatives
had the highest activity. Xu et al. then combined ligustrazine
-birch acid with amino acids and
dipeptides, which raised its antitumor activity compared to unmodified ligustrazine
-
betulinic acid [16].

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Activation of the mitochondria-mediated endogenous apoptotic pathway decreases the
mitochondrial membrane potential, which leads to a significant increase in mitochondrial membrane
permeability. Then it causes the release of cytochrome c. Cytochrome c is released into the cytosol and
activates a family of caspases. Then a series of cascade reactions are triggered [17–19]. Studies have
shown that the increased production of reactive oxygen species (ROS) also induces a decrease in the
mitochondrial membrane potential, followed by the release of cytochrome c, which ultimately leads to
apoptosis [20,21]. In addition, ROS induce activation of the JNK signaling pathway, which is a key
mediator of apoptosis [22,23].
In this study, we synthesized a series of novel amide anthraquinone-amino acid derivatives using
2-methylanthraquinone as a raw material. We carried out structural modifications at the C-1 position,
and combined several different amino acids at the C-2 position. All newly synthesized compounds
were identified by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy. The most
active compounds were screened by MTT assay and their half-maximal inhibitory concentration (IC50)
values were determined. We then established an optimal model a three-dimensional quantitative
structure activity relationship (3D-QSAR) to correlate biological activity with the chemical structure of
-
the synthetic series. Compound 8a showed good antitumor activity, inducing significant apoptosis via
JNK activation in HCT116 cancer cells.
2. Results
2.1. Chemistry
A series of novel anthraquinone
-amino acid derivatives 8a–8h were synthesized following the
general procedures outlined in Scheme 1.
Scheme 1. Synthesis of anthraquinone-amino acid derivatives.
Initially, 2
-
methylanthraquinone was prepared as a starting material. Subsequently, 2
-
methyl
methyl
anthraquinone was subjected to nitration under acidic conditions to form 1
anthraquinone. This compound was then oxidized to form 1 nitro
addition of Na2Cr2O7. This reaction product was further subjected to acylation to form 1
anthraquinone. Finally, a series of 1 nitro acyl anthraquinone amino acids 8a 8h were synthesized
-nitro
-
2
-
-
-
2
-carboxy anthraquinone by the
-
nitro benzoyl
-2-
-
-2
-
-
–
by reactions with several different amino acids, including leucine, valine, phenylalanine, glycine,
proline, alanine, methionine and glutamic, respectively.

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2.2. Compound 8a Inhibits Tumor Cell Proliferation But Does Not Affect Normal Cells
The effects of the synthesized amide anthraquinone derivatives on the proliferative activity of
eight human cancerous cell lines (EC9706, MCF 7, SGC-7901, HepG2, QBC939, HeLa, HCT116 and
-
SW480) in addition to two normal human cell lines (colorectal FHC and liver HL7702) were evaluated
using the standard MTT assay. The median IC50 values for each compound are presented in Table 1.
Table 1. IC50 values [a] (µg/mL) of compounds.
IC50 (µg/mL)
Compounds
8a
8b
8c
8d
8e
8f
8g
8h
17.80 ±
21.01 ±
30.12 ±
27.75 ±
35.03 ±
98.43 ±
86.49 ±
100.38 ±
HCT116 [b]
MCF-7 [c]
EC9708 [d]
QBC939 [e]
SGC-7901 [f]
HeLa [g]
0.03
0.38
0.02
0.11
0.25
0.13
0.02
0.04
33.34 ±
0.28
30.81 ±
0.05
28.26 ±
0.13
26.87 ±
0.06
31.31 ±
0.13
342.19 ±
0.22
275.52 ±
0.07
546.68 ±
0.31
44.00 ±
38.94 ±
30.24 ±
41.47 ±
47.35 ±
257.31 ±
300.18 ±
112.41 ±
0.61
0.02
0.23
0.14
0.21
0.20
0.11
0.03
43.13 ±
0.05
72.88 ±
0.16
34.25 ±
0.42
36.71 ±
0.04
37.73 ±
0.23
357.52 ±
0.08
290.94 ±
0.05
112.02 ±
0.03
55.82 ±
67.98 ±
61.60 ±
60.83 ±
72.31 ±
165.34 ±
235.81 ±
200.35 ±
0.16
0.18
0.14
0.12
0.33
0.21
0.02
0.05
29.75 ±
0.15
27.87 ±
0.22
25.94 ±
0.22
28.70 ±
0.17
48.82 ±
0.13
241.31 ±
0.13
122.52 ±
0.05
334.28 ±
0.04
31.09 ±
42.36 ±
39.50 ±
43.08 ±
60.49 ±
155.38 ±
158.37 ±
119.93 ±
SW480 [h]
HepG2 [i]
0.10
0.68
0.31
0.05
0.07
0.14
0.04
0.03
44.65 ±
0.03
>1000
43.96 ±
0.53
>1000
58.34 ±
0.22
>1000
40.11 ±
0.02
>1000
49.07 ±
0.08
>1000
296.42 ±
0.21
>1000
520.52 ±
0.22
467.74 ±
0.06
>1000
HL-7702 [j]
FHC [k]
[a]
>1000
>1000
[b,h]
>1000
>1000
>1000
>1000
>1000
>1000
>1000
The half
-
maximal inhibitory concentration (IC50) of cells treated with the compounds 48 h.
Colon cancer,
[f]
[i]
Breast cancer, [d] Esophageal cancer, [e] Cholangiocarcinoma, Gastric cancer, [g] Cervical cancer, Liver cancer,
Normal human hepatocyte, [k] Normal human colorectal mucosa.
[c]
[j]
Some of the compounds 8a
cells, with IC50 values ranging from 21.01 to 72.88
liver cells (HL7702) and normal colorectal cells (FHC). It is worth noting that compound 8a showed
the greatest inhibition of HCT116 cells proliferation with an IC50 of 17.80 g/mL. In addition, the
IC50 of compound 8a for SGC-7901 cells was 55.82 g/mL, which was higher than that for the other
tumor cells, indicating that this cell line was less sensitive to compound 8a than the other tumor cell
lines. The IC50 of 8b on HCT116 was 21.01 g/mL, indicating that compound 8b also exerted strong
–8h significantly inhibited the proliferative capacity of the tested tumor
µg/mL, all while exerting a minimal effect on normal
µ
µ
µ
proliferation suppression activity on this cell line. In contrast, QBC939 cells were less sensitive to
compound 8b than the other tumor cells lines, with an IC50 of 72.88 µg/mL. SGC-7901 cells were also
less sensitive to 8c, but this compound showed higher suppression activity in HeLa cells, with an IC50
of 25.94
was less suppression activity to SGC-7901 cells. Compound 8e had the weakest effect on SGC-7901 with
an IC50 of 60.49 g/mL. Finally, compounds 8f, 8g, and 8h all significantly inhibited the proliferation
of HCT116 cells. These data indicate the activity of the anthraquinone derivative with leucine as the
-substituent on the anthraquinone ring is superior to that of anthraquinone derivatives with other
amino acid.
Figure 2A shows the parent compound structure. Compared with the structure of the rhein
µg/mL. MCF-7 was more sensitive to compound 8d than other cells, although this compound
µ
β
valine [24], the hydroxyl group at position 8 is removed, the hydroxyl group at the position 1 is replaced
with a nitro group, and the amino acid group at the meta position is transferred to the ortho position.
In addition, there was a significant difference in inhibitory activity of compounds with substitution of
the same amino acid at different sites. The rhein valine IC50 was 33.51 µg/mL, while the IC50 of 8b was

Molecules 2020, 25, 1672
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significantly enhanced at 27.87
µg/mL (Figure 2B,C). The nitro group is a strong electron withdrawing
group, while the hydroxyl group is an electron donating group.
Figure 2.
) the target compound;
(D) 1-nitro-2-acyl-anthraquinone valine (8b).
Comparison of rhein and
1
-
nitro
-
2
-
acyl
-
anthraquinone valine structures.
) rhein valine;
(
A
(
B) 1-nitro-2-acyl-anthraquinone leucine (8a); (C
2.3. 3D-QSAR
To better understand the structure-function relationship, we conducted a 3D-QSAR study to a
series of amide anthraquinone derivatives using CoMFA and CoMSIA. The pIC50 values ( log IC50) of
−
HCT116 cells were selected for 3D-QSAR analysis according to the results of the MTT assay of these
compounds, which were found to be superior to the other cell types tested (Table 1). The parameters of
the CoMFA and CoMSIA models indicated good relevance and predictive ability (CoMFA: R2 = 0.965,
q2 = 0.525, two components; CoMSIA: R2 = 0.983, q2 = 0.6, two components); the contribution of the
steric and electrostatic fields was 80: 20. The predicted and experimental values obtained for the best
CoMFA and CoMSIA models for each compound are shown in Table 2.
The correlation between the training-set proliferation suppression activity against HCT116 cells
is shown in Figure 3. The results indicated good predictability of the molecules under investigation.
In summary, the parameters of this CoMFA and CoMSIA models were found to be reliable and to
provide a credible guarantee that novel amide anthraquinone compounds with strong anti-tumor
activity and few side-effects can be easily designed.

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Table 2. Experimental and predicted values of pIC50 obtained with the best CoMFA and CoMSIA
models, in HCT116 cells.
CoMFA Model
CoMSIA Model
IC50
µg/mL
Experimental
pIC50
QSAR
Set
Compound
Predicted
Predicted
Residual
Residual
pIC50
pIC50
8a
8b
8c
8d
8e
8f
training
training
training
training
training
training
training
training
17.80
21.01
30.12
27.75
35.03
98.43
86.49
100.38
−1.25
−1.32
−1.48
−1.44
−1.54
−1.99
−1.94
−2.00
−1.283
−1.287
−1.435
−1.421
−1.524
−2.009
−1.976
−2.023
0.033
−0.033
−0.045
0.019
−1.262
−1.230
−1.577
−1.593
−1.536
−1.974
−2.054
−1.787
0.033
−0.033
0.097
0.153
−0.004
0.019
0.036
0.023
−0.016
0.019
8g
8h
0.036
0.023
Figure 3. The training-set corresponding to the predicted value (diamonds) pIC50 using the CoMFA
(A) and CoMSIA (B) model, in HCT116 cells.
Figure 4 shows a three-dimensional equation of the stereo and electrostatic fields of a CoMFA
model with 8a as the template. As shown in Figure 4A, the CoMFA stereograph shows that the
position of the terminal carboxyl group at the side-chain of the anthraquinone ring in compound
8a is at a favorable position. This indicates that the introduction of carboxyl groups at this position
significantly increases biological activity. In addition, the CoMFA electrostatic diagram shown in
Figure 4B demonstrates that the carboxyl group in compound 8a is close to the blue region, which is a
desirable trait for increasing the antiproliferative activity. The red regions around the nitrogen atoms
in 8a indicate that it also contributes to antiproliferation activity against tumor cells by increasing the
negative charge of the compound.
Figure 4. The three-dimensional equipotential map of the CoMFA stereo electrostatic field.
(A) Three-dimensional equipotential map; (B) Electrostatic equipotential map.
Figure 5 also shows a three-dimensional equation of the stereo and electrostatic fields of a CoMFA
model with 8a as the template. In this CoMFA model (Figure 5A), the C-terminal in the leucine side-chain

Molecules 2020, 25, 1672
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and on the left of the anthraquinone ring has yellow areas, which indicate that the introduction of
small molecular groups in this region can increase the antiproliferative activity. The CoMSIA model
electrostatic field equipotential map illustrated in Figure 5B shows the anthraquinone ring and leucine
side-chain in compound 8a surrounded by a blue equipotential domain, which is a desirable trait for
increasing antiproliferative activity.
Figure 5. The three-dimensional equipotential map of the CoMSIA model. (A) Stereo equipotential
map; (B) Electrostatic equipotential map; (C) Hydrophobic equipotential map; (D) H-bond combined
equipotential map.
For the hydrophobic CoMSIA model, the yellow in Figure 5C,D represents the introduction of a
hydrophobic group to facilitate an increase in the inhibitory activity of the compound, while the white
region represents the introduction of a hydrophobic group, which reduces the inhibitory activity of the
compound. The hydrophobic field map shown in Figure 5C demonstrates that the nitro group of the
anthraquinone ring and the left side-chain of the anthraquinone ring have white regions, indicating that
this group can enhance the hydrophobicity of the molecule and further enhance the antiproliferative
activity of the compound. Figure 5D shows a hydrogen bond acceptor field for the CoMSIA model. It
can be seen that the nitro group of the anthraquinone ring and the left side-chain of the anthraquinone
ring are surrounded by a magenta region, with only a small red region around the anthracene ring,
indicating that the hydrogen bond acceptor group at this position is advantageous.
2.4. 8a Induces Apoptosis in HCT116 Cells
Natural anthraquinones, such as rubiadin, physcion, emodin and soranjidiol [25–27], have been
reported to induce apoptosis in tumor cells. Therefore, after detecting the effect of 8a on tumor
cell proliferation, we investigated correlation between the anticancer activity of compound 8a and
apoptosis. The effects of compound 8a treatment on HCT116 cell apoptosis were detected by flow
cytometry following Annexin V/PI staining. Treatment with compound 8a at different concentrations
(0, 10, 20, and 40 µg/mL) for 24 h significantly increased the rate of apoptosis (11.24%, 18.97%, and
26.94%, respectively) compared with that in the control group (0.06%) (Figure 6A,B). This indicated
that the mechanism by which 8a inhibits cell proliferation may associate with apoptosis.

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Figure 6. Analysis of HCT116 cell line apoptosis following treatment with 8a
cytometric analysis of HCT116 cell apoptosis following treatment with different concentrations of 8a
) A histogram of the number of apoptotic cells after 8a treatment. Data represent the mean standard
deviation of three independent experiments * p < 0.05, compared with control group.
.
(A) Flow
;
(B
±
In addition, the expression of apoptosis-related proteins after 8a treatment was detected by
western blot. Previous studies have shown that the Bcl 2 protein family is one of the most interesting
oncogene-related proteins in programmed cell death [28]. Therefore, we analyzed the levels of
Bcl 2, Bcl xL and Bax after 24 h of treatment with different concentrations (0, 10, 20, and 40 g/mL)
8a. The results showed that the levels of Bcl 2 and Bcl xL gradually decreased with increasing 8a
concentration, whereas Bax expression was increased. Moreover, the phosphorylation levels of Bcl
and Bcl xL increased with increasing 8a concentration. These results indicated that 8a activated Bax
expression and inhibited the expression of Bcl-2 and Bcl xL protein (Figure 7A,C). This suggested that
-
-
-
µ
-
-
-
2
-
-
8a may induce apoptosis in HCT116 cells by regulating Bcl-2 family proteins.
Caspases represent a key molecular group in the regulation of apoptosis. In caspases, there is an
upstream and downstream relationship between the initiator of apoptosis and executioner. Caspase 3
is one of the key executers of apoptosis, as it is either partially or totally responsible for the proteolytic
cleavage of many key proteins. For example, when caspase 9 is activated, it activates caspase 3, which
ultimately triggers cell apoptosis [29,30]. After detecting the expression of the corresponding Bcl-2
family proteins after 8a treatment by western blotting, we went on to detect the expression levels of
PARP, caspase 9, cleaved caspase 9, caspase 3, and cleaved caspase 3 proteins. As shown in Figure 7B,D,
caspase 9 and caspase 3 expression levels in HCT116 cells gradually decreased with increasing of
concentrations of in 8a, while the expression of cleaved caspase 9 and cleaved caspase 3 increased in a
dose-dependent manner. zVAD-FMK is a pan
binds irreversibly to the catalytic site of caspase proteases, thereby inhibiting apoptosis. As shown in
Figure 7E, HCT116 cells were co incubated with 8a (20 g/mL) and zVAD FMK (5 mM) for 24 h, after
which cell viability was measured by MTT assay. After 24 h of treatment with zVAD FMK, cell viability
is 93%. However, co incubation with 8a and zVAD-FMK, cell viability increased significantly from 38%
-caspase inhibitor that penetrates cell membranes and
-
µ
-
-
-
of the effect observed following treatment with 8a to 55%. These results indicated that caspases are
activated by 8a.

Molecules 2020, 25, 1672
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Figure 7. Effect of 8a on apoptosis
with 20 g/mL 8a for different time
caspase 9, Bcl 2, phosphorylated Bcl
western blot analysis; β-actin was used as a loading control in each lane. Representative data of three
independent experiments are shown; ( ) and ( ) Grayscale analysis of the relative expression of
apoptosis related proteins detected by Western blotting. Data represent the mean standard deviation
of three independent experiments. ** p < 0.01, * p < 0.05; compared with control group. ( ) Cells were
exposed to 8a (20 g/mL) and/or zVAD FMK (5 mM) for 24 h, and cell viability was measured by MTT
standard deviation of three independent experiments * p < 0.05,
-
related protein expression. (
periods. PARP, caspase 3, cleaved caspase 3, caspase 9, cleaved
2, Bcl XL, phosphorylated Bcl xL and Bax were detected by
A) and (B) HCT116 cells were treated
µ
-
-
-
-
-
C
D
-
±
E
µ
-
assay. Data represent the mean
** p < 0.01.
±
2.5. Compound 8a Induces Generation of ROS and Affects Mitochondrial Membrane Potential
Intracellular ROS are key factors in the activation of many signaling pathways, involved in a
variety of biological functions including apoptosis and necrosis. High levels of ROS are capable of
inducing tumor cell apoptosis [31]. We evaluated the ability of the compounds to induce ROS by
flow cytometric analysis of the fluorescent probe DCFH
-DA. Compound 8a was found to increase
ROS levels in a dose dependent manner compared to the 0.01% DMSO
-
-
treated control (Figure 8A,B),
indicating that 8a induces the production of intracellular ROS. Next, we investigated ROS production
before and after induction with 8a and NAC (N-acetyl-L-cysteine). The results indicated that NAC
prevents 8a-induced ROS production (Figure 8C,D).
Decreased mitochondrial membrane potential is a hallmark of early cell apoptosis, and a decrease
in mitochondrial membrane potential leads to the release of certain mitochondrial proteins, such as
cytochrome c, which mediate apoptosis. Therefore, we examined changes in mitochondrial membrane
potential by flow cytometry. HCT116 cells were treated with 8a (10 or 20
µg/mL) for 24 h and then stained
with 2 g/mL JC 1 for 30 min prior to flow cytometric analysis. Green /orange fluorescence ratio of JC-1,
µ
-
compare with control, is 200% and 494.4%, respectively (Figure 8E,F). The results showed compound
8a decreased the mitochondrial membrane potential in a dose-dependent manner. During apoptosis,
mitochondrial cytochrome C is released into the cytoplasm and triggers the activation cascade of
caspase. HCT116 cells were collected after 8a treatment, cytoplasmic protein and mitochondrial protein
were extracted. The levels of cytochrome c in the cytoplasm and mitochondria were detected by
Western blotting. After treatment of HCT116 cells with 8a (10 or 20 µg/mL) for 24 h, cytoplasmic levels

Molecules 2020, 25, 1672
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of cytochrome c increased, while the levels in the mitochondria decreased. The results indicated that
compound 8a reduces the mitochondrial membrane potential, thus implicating the mitochondria in
8a-induced apoptosis (Figure 8G,H).
Figure 8. Treatment with 8a induced ROS production and mitochondria
) ROS production by HCT116 cells treated with 10 or 20 g/mL 8a for 24 h; 0,01% DMSO was used
as the control; ( ) ROS levels were determined by flow cytometry after treatment of HCT116 cells
with 20 g/mL 8a with or without NAC and stained with DCFH DA; ( ) and ( ) Mean fluorescence
is expressed as a percentage of ROS; ( ) Mitochondrial membrane potential (JC 1 fluorescence) of
cells was reduced after treatment with different concentrations of 8a at 37 ◦C for 24 h; (
) Changes in
mitochondrial membrane potential (MMP) after 8a treatment; ( ) HCT116 cells were treated with 10 or
20 g/mL 8a for 24 h, and the levels of cytochrome c in the mitochondria and cytoplasm were determined
by western blot analysis, COX IV and β-Actin were used as controls, respectively; ( ) Grayscale
analysis of the relative protein levels of mitochondrial and cytoplasmic cytochrome c. Data represent
the mean standard deviation of three independent experiments. * p < 0.05, ** p < 0.01. Among them,
Figure B, F, H compared with the control group.
-associated protein expression.
(
A
µ
C
µ
-
B
D
E
-
F
G
µ
-
H
±

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2.6. Compound 8a-induced ROS Production Is Associated with JNK Activation
In the ROS-induced signaling pathway, JNK is an important downstream molecule that activates
apoptosis. ROS induces JNK activation, which in turn induces the expression of pro-apoptotic proteins
in tumor cells to activate apoptosis via the mitochondrial pathway [32,33]. JNK is a member of the
mitogen-activated protein kinase family and is capable of phosphorylating and activating c-Jun. JNK is
involved in regulation of biological processes, such as cell proliferating, cell differentiation, cell death
and stress responses, and further promotes expression of p53, Bax, FasL and tumor necrosis factor by
promoting the activity of transcription factor complex activator protein-1.
To further elucidate the mechanism by which 8a inhibits proliferation and induces apoptosis of
HCT116 cells, the levels of phosphorylated and total JNK protein were examined by western blot analysis.
As shown in Figure 9A, total JNK levels were not altered by a treatment with compound 8a, while the
levels of phosphorylated JNK gradually increased with time. Phosphorylation of JNK activated by the
ROS pathway results in overexpression of tumor suppressor factors, thereby inducing apoptosis [34].
In addition, JNK phosphorylation is associated with ROS production [35,36]. To determine whether
ROS production contributes to JNK1/2 phosphorylation and its related mechanisms, we blocked
8a-induced ROS production in HCT116 cells and then analyzed the changes in JNK 1/2 phosphorylation
by western blotting. As show in the Figure 9B,C, the results show that the levels of p-JNK after
treatment of HCT-116 cells with NAC were on the level of control samples. Furthermore, pre-treatment
of cells with NAC reduced the levels of p-JNK induced by 8a. These results indicate that compound
8a-induced ROS production is associated with JNK activation.
Figure 9. The effect of 8a treatment on phosphorylated JNK and JNK expression levels. (
analysis of phosphorylated JNK and JNK expression levels of in HCT116 cells treated with 20
A
) Western blot
g/mL
JNK proteins were determined after
µ
8a for time
-
periods; (
B
) The expression levels of the phospho
-
treatment of HCT116 cells with 20
µ
g/mL 8a with or without NAC. Data represent the mean
±
standard
deviation of three independent experiments; (
phosphorylated JNK. Data represent the mean
* p < 0.05; ( ) MTT assay of HCT116 cells viability following treatment with 8a and SP600125 for 24 h.
C
) Grayscale analysis of the relative protein level of
±
standard deviation of three independent experiments.
D
Furthermore, to further investigate whether activation of phosphorylated JNK affects cell viability,
HCT116 was co treated with 8a and SP600125 (JNK inhibitor), and the effect of phosphorylated JNK
inactivation on HCT116 cell activity was examined by MTT. Compared with the control, HCT116 cell
-

Molecules 2020, 25, 1672
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viability was significantly inhibited (39%) following exposure to 8a (Figure 9D). Following co
-
treatment
with 8a and SP600126, the rate cell viability was 55%. The inhibitory proliferation activty of 8a were
partially weakened by the usage of 5 M Sp600125. The results showed that the increase of the
µ
expression of phosphorylated JNK after 8a treated will decrease the cell viability. It also indicated that
the increase of the expression of phosphorylated JNK will inhibit cell proliferation, which is related.
3. Discussion
Natural anthraquinone compounds have been shown to exert anticancer effects both in vivo
and in vitro
[37]. Anthraquinone compounds induce cell death in a variety of cancer cell lines,
including LS1034, HepG2, MCF
-
7, MDA MB 231, MCF 10A, SW480, and SW620 cells [38 39]. Chemical
-
-
-
,
modifications of the side-chain represent an effective approach to improving the activity of certain
anticancer drugs.
Most of all the compounds inhibited cancer cell proliferation according to the MTT assay.
Compounds in which the side-chain groups were replaced by leucine exhibited higher activity.
Therefore, it is shown that 8a has significant anti-proliferative activity on a variety of tested cells, and
the effect on HCT116 cells is more significant. Furthermore, 8a induced HCT116 cell line death via
apoptosis in a time-and dose-dependent manners.
Anthraquinone induces the generation of ROS [40], which induce apoptosis of tumor cells via
JNK activation [41,42]. Activated JNK phosphorylates Bcl-2 [43]. The production of ROS causes JNK
activation and induces the death of various cell lines, such as human pancreatic cancer cells, Sertoli
cells, and human cervical cancer cells [44]. Extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun
N-terminal kinase (JNK), and p38 MAPK are the most widely studied members of the MAPK family [45].
Our data demonstrated that 8a induced HCT116 cell apoptosis via the ROS/JNK signaling pathway.
This finding suggests that ROS acts an upstream signaling molecule in the pathway. Treatment of
HCT116 cells with 8a increased ROS levels in a dose-dependent manner. In addition, inhibition of
ROS generation by NAC (5 mM) markedly reduced activation of JNK in HCT116 cells, suggesting that
8a induces HCT116 cell death, at least partially, through ROS-mediated activation of JNK. However,
NAC only partially blocks 8a induced ROS production (Figure 8D). Some studies have shown that
NAC has a concentration-dependent inhibitory effect on ROS induction [46]. Moreover, other studies
have shown that NAC is a weak reducing agent and a poorer antioxidant compared to glutathione
(reduced form) (GSH) [47], so the experimental results show that NAC partially blocks 8a-induced
ROS production. Moreover, we also concluded that although we have performed many preliminary
experiments in the previous period, the concentration of 5 mM NAC still cannot quickly clear the ROS
induced by 8a. In addition, JNK inhibition only partially rescue the anti-proliferative effects induced
by compound 8a (Figure 9E), we concluded that 8a inhibition of HCT1116 cell proliferation may also
be related to the regulation of other signaling pathways.
In addition, using 3D-QSAR analysis, results indicated that if the hydrophilic target protein is
near to the 8a leucine side chain, it may increase antitumor activity and further induce apoptosis.
In the next, it is an interesting and worthy further study of 8a-induced apoptosis signaling pathways.
The reduction in Bcl
studies have shown that phosphorylation of Bcl
oligomerization in the mitochondria and cytochrome c release in the cytoplasm [22
-
2/Bax ratio was determined as a marker of an apoptosis signaling [48]. Numerous
xL(S62), but not Bcl 2, induces cell apoptosis via Bax
49]. However, the
-
-
,
results showed that both Bcl-2 and Bcl-xL were phosphorylated after treatment with 8a, therefore, the
specific mechanism of 8a-induced HCT116 cell apoptosis needs further study.
4. Materials and Methods
4.1. Reagents
The following reagents and equipment were used: analytically pure (AR) 2
(Chemical & Technology Company, Shanghai, China); SHZ-C type circulating water multi
-
methylanthraquinone
purpose
-

Molecules 2020, 25, 1672
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vacuum pump (Gongyi City Yingyu Yuhua Instrument Factory, Henan, China), RE-52A rotary
evaporator (Yarong Biochemical Instrument Factory, Shanghai, China); X-4 digital display micro-melting
point tester (Beijing Tektronix Instrument Company, Beijing, China), HP8453 UV-Vis absorption
spectrometer (Hewlett Packard, California, The United States); DRX300MHZ nuclear magnetic
resonance instrument (Bruker, Worcester, Switzerland); RPMI-1640 medium (HyClone, Logan,
Utah, The United States); fetal bovine serum (Sangon Biotechnology, Shanghai, China); MTT
(Sigma, Maryland, The United States); Annexin V-FITC assay kit (Pharmingen Becton Dickinson,
New York, The United States); anti-Bcl-2, anti-phosphorylated Bcl-2 (Bioss, city, China); anti-Bcl-XL,
anti-phosphorylated Bcl-XL, anti-Bax, anti-Caspase-3, anti-Caspase-9, anti-cleaved caspase-3,
anti-cleaved caspase-9, anti-PARP antibody, anti-phosphorylated (Thr183/Tyr185) JNK, anti-JNK,
ROS detection kit, N-Acetyl-L-cysteine (NAC) and mitochondrial membrane potential detection kit
(Beyotime Biotechnology, Shanghai, China); anti-cytochrome c, anti-COX-IV antibody, horseradish
peroxidase-labeled goat anti-rabbit IgG secondary antibody, anti- -actin antibody (Bioworld,
β
Minnesota, The United States); ECL assay kit (Engreen Biosystem, Beijing, China); PVDF membrane
(Millipore, Massachusetts, Germany); BCA protein quantification kit (Minbio, Shanxi, China).
4.2. Compounds
We used 2-methylanthraquinone as a raw material to synthesize the 1-nitro-2-benzoyl
chloride
acylation.
anthraquinone
The 1-nitro-2-acylanthraquinone-amino acid derivatives 8a
The characterization results of
intermediate
by
sequential
nitration,
oxidation,
and
–
8h were then
synthesized by reaction with suitable amino acids.
N-[(9,10-dihydro-1-nitro-9,10-dioxo-2-anthracenyl)carbonyl-leucine] (8a) are as follows:
IR,
v/cm-1: 3487 (w), 3371 (w), 3500–2500 (w), 2960 (w), 1724 (m), 1680 (s), 1658 (s), 1589 (m), 1552
1
(s), 1469 (w), 1411 (w), 1369 (w), 1319 (m), 1280 (s), 711 (m); H-NMR (300 MHz, DMSO),
δ ppm:
0.85–0.91 (m, 6H), 1.55–1.70 (m, 3H), 4.34 (s, 1H), 7.91–7.94 (m, 2H), 8.01–8.20 (m, 3H), 8.44–8.47 (d,
1H), 9.29–9.31 (d, 1H), 12.82 (s, 1H); 13C-NMR (1200 MHz, DMSO),
ppm: 23.6, 25.5, 26.9, 42.3, 53.4,
129.4, 131.4, 135.1, 135.6, 136.7, 137.5, 138.0, 148.5, 165.7, 175.9, 182.4, 183.1; UV max (THF) (nm):
δ
λ
261, 326. The characterization results of the other anthraquinone derivatives 8b–8h are given in the
Supplementary Materials.
4.3. Cell Culture
The human esophageal cancer cell line EC9706 was obtained from Cancer Hospital and Institute
by Prof. Ming Rong Wang, Chinese Academy Medical of Sciences. The human colorectal carcinoma cell
lines HCT116 and SW480, hepatoma cell line HepG2, cervical carcinoma cell line HeLa, breast cancer
cell line MCF-7, gastric cancer cell line SGC-7901, cholangiocarcinoma cell line QBC939, normal fetal
colon cell line FHC and liver cell line HL-7702 were obtained from the Cell Bank of the Shanghai Chinese
Academy of Sciences. All cells were cultured in RPMI 1640 medium containing 100 U/mL penicillin
and streptomycin and 10% fetal bovine serum and maintained at 37◦C under a 5% CO2 atmosphere.
4.4. In Vitro Cell Proliferation Assays
We analyzed the inhibitory effects of eight anthraquinone-amino acid derivatives on the
proliferation of eight tumor cell lines and two normal cell lines in vitro using MTT assays. Cells were
added to a 96-well plate with (5,000 cells per well in 100
with different concentrations (10–100 g/mL) of 8a 8h for 48 h and then incubated with 20
(5 mg/mL) for another 4 h at 37◦C. DMSO (150
L) was then added to each well to dissolve the formazan
µL RPMI-1640 medium). Cells were treated
µ
–
µL MTT
µ
crystals. The absorbance of each well was read at 490 nm on a microtiter plate ELISA reader. The mean
values were obtained from a minimum of three parallel experiments. IC50 values were calculated.

Molecules 2020, 25, 1672
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4.5. 3D-QSAR Study
The amide anthraquinone derivatives (8a
–
8h) were subjected to 3D-QSAR studies using
Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis
(CoMSIA), which showed inhibition of the proliferative activity of colon cancer cell lines. Since the
antitumor activity of 8a was the highest, the structure of Compound 8a was used as a representative
for analysis. The CoMFA and CoMSIA models were generated using the SYBYL-X 2.0 QSAR software
module, in which the negative logarithm of the IC (pIC50) was used as the modeling response value.
The IC50 value was measured in terms of colon cancer cell proliferation. Structural changes (CoMFA
fields) were correlated with changes in the antiproliferative activity of the amide anthraquinone
derivatives using partial least squares regression (PLS).
4.6. Apoptotic Assay
HCT116 cells were added to a 6-well plate (5
with 8a for 24 h. Then, the cells were collected and centrifuged (10,000 g
once with 1 mL of PBS. Cells were suspended in 500 L binding buffer (containing 5
V-FITC and 1 L propidium iodide [PI]) and incubated for 30 min in the dark. Apoptosis cells were
×
106/well). After attachment, the cells were treated
5 min). Cells were washed
L Annexin
×
µ
µ
µ
detected by flow cytometry (FACSCalibur, BD, The United States).
4.7. Detection of ROS by DCFH-DA
Cells were added to a 6-well plate (1
×
104/well) and incubated for 24 h to allow adherence. Set
0.1% DMSO as the control group, 8a as the treatment group, NAC as the treatment group, 8a + NAC
as the treatment group. Among them, in the 8a + NAC treatment group, 8a was added after 5 mM
NAC treatment for 1 h. Cells that had been treated for 24 h were collected. The cells were then washed
three times with PBS and incubated with 10
analyzed by flow cytometry.
µM DCFH-DA for 30 min in the dark. ROS levels were
4.8. Determination of Mitochondrial Membrane Potential
The 1
104 cells were treated with different concentrations of 8a for 24 h. Cells were then incubated
with 500
L JC-1 (10 mg/mL) for 30 min at 37 ◦C in the dark. The level of mitochondrial membrane
×
µ
potential depolarization was measured by flow cytometry. Data were analyzed using the Cell Quest
program (BD, New York, The United States).
4.9. Western Blot Analysis
The expression of signaling pathway-related proteins was detected by western blot analysis. Cell
(1
the same way as in step 4.7. Resuspend the cells in Buffer A (1 mM EDTA, 1 mM PMSF, 0.28
apotinin, 50 g/mL leupeptin, 7 g/mL pepstain A) and transferred to a 1 mL glass homogenizer and
pull up and down 30 times in an ice bath. Cells were collected and centrifuged (1000 g
10 min, 4 ◦C).
The obtained supernatant and precipitate were the crude cytoplasm and mitochondria, respectively.
The cytosolic fraction was transferred to an ultra-ion tube and centrifuged (100,000g
1 hr, 4 ◦C), and
the resulting supernatant was the purified cytosolic fraction. The mitochondrial pellet was resuspended
in Buffer B (1mM EGTA, 1mM PMSF, 0.28 g/mL apotinin, 50 g/mL leupeptin, 7 g/mL pepstain
A), centrifuged (10,000g
10 min, 4 ◦C), and repeated 3 times. The mitochondria pellet was lysed in
cell lysate buffer (1% NP-40, 2 g/mL aprotinin, 2 g/mL leupeptin, 1mM EDTA, 1mM Na vanadate).
×
106) were plated in 90-mm dishes and treated with 8a for 24 h. The cells were treated with NAC in
µg/mL
µ
µ
×
×
µ
µ
µ
×
µ
µ
Protein concentrations in the supernatant were determined by using a Minbio BCA protein assay kit.
Protein were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was
blocked, and incubated with primary detection antibodies overnight at 4 ◦C. After washing three times
with TBS-T, the membranes were incubated with the secondary detection antibody. After washing
three times with TBS-T, protein bands were visualized using an enhanced chemoluminescence (ECL)

Molecules 2020, 25, 1672
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kit (luminol/enhancer solution, peroxide solution, protein detection complex). Next, in the gel imaging
system, the protein bands were scanned and the pictures were imported into the Image J software for
gray analysis.
We have adopted two analysis methods:
Relative protein level/fold = Experimental group gray value/ Control group gray value
Ratio of relative intensity of control = Experimental group relative protein level/Control
relative protein level
4.10. Statistical Analysis
All data in this paper were performed in three independent experiments. The SPSS 11.5 software
(company, city, state abbrev if USA, country) was used to calculate IC50 values. Differences among
groups were assessed using one-way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01 indicates a
statistically significant difference.
5. Conclusions
In summary, we have generated a series of new anthraquinones, compounds 8a-8h, by structural
modification of anthraquinone, an active ingredient of several traditional Chinese medicines. Among the
compounds generated, our study showed that 8a has the best anti-tumor activity against colon cancer
cells and induces apoptosis via the ROS/JNK signaling pathways. In 8a-induced apoptosis, initial
activation of the ROS-JNK signaling pathway caused increased ROS production, JNK phosphorylation,
followed by a decrease in mitochondrial membrane potential and release of cytochrome c mediated
by the actions of Bax and Bcl
turn leads to activation of the caspase signaling pathway, and ultimately to apoptosis (Figure 10).
Moreover, by analyzing structure activity relationships, we have demonstrated that the solubility and
biological activity of the compound synthesized can be greatly improved by increasing the electron
withdrawing capacity of the nitro group at the C 1 position and substitution of the amino acid at the
position. Thus, our study provides new insights for the future design and development of more
effective anti-tumor drugs.
-2. These results lead to caspase 9 and caspase 3 cleavage, which in
-
-
β
Figure 10. Summary of the proposed mechanism by which 8a induces apoptosis. Reactive oxygen
species(ROS); C Jun N terminal kinase (JNK); N-Acetyl-cysteine (NAC); Apoptosis regulator Bcl-2
(Bcl-2); Bcl-2 Associated X Protein (BAX); Poly ADP-ribose polymerase (PARP).

Molecules 2020, 25, 1672
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Supplementary Materials: The following are available online, Figure S1: The 1HNMR (300 MHz) spectrum of
compound 8a, Figure S2: The 13CNMR (300 MHz) spectrum of compound 8a, Figure S3: The 1HNMR (300 MHz)
spectrum of compound 8b, Figure S4: The 13 CNMR (300 MHz) spectrum of compound 8b, Figure S5: The 1HNMR
(300 MHz) spectrum of compound 8c, Figure S6: The 1HNMR (300 MHz) spectrum of compound 8d, Figure S7:
The 13CNMR (300 MHz) spectrum of compound 8d, Figure S8: The 1HNMR (300 MHz) spectrum of compound 8e
,
Figure S9: The 13CNMR (300 MHz) spectrum of compound 8e, Figure S10: The 1HNMR (300 MHz) spectrum of
compound 8f, Figure S11: The 1HNMR (300 MHz) spectrum of compound 8g, Figure S12: The 1HNMR (300 MHz)
spectrum of compound 8h.
Author Contributions: Y.L. and L.Z. conceived and designed the experiment; F.G. and Y.G. performed the
experiments, analyzed the data, and wrote the paper; K.M., L.Z., Q.S. and L.F. participated in the synthesis of
target compounds; T.C., Z.W., Y.L. and Y.Z. contributed analysis tools and helped in MS and QSAR analysis; All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by Transformation of Scientific and Technological Achievements Programs
of Higher Education Institutions in Shanxi (TSTAP), Key projects of Shanxi Province (No. 201903D321095), and
Research on Key Technologies of Modernization of Traditional Chinese Medicine in Shanxi Province Zhendong
Special Foundation (No. 2016ZD0502). We thank Prof. Daxiong Han from Medical College Xiamen University for
software support.
Conflicts of Interest: The authors declare they have no conflict of interest.
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Sample Availability: Samples of the compounds 8a and 8d are available from the authors.
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