Two novel camptothecin derivatives inhibit colorectal cancer proliferation via induction of cell cycle arrest and apoptosis in vitro and in vivo

Article abstract of DOI:10.1016/j.ejps.2018.08.018

At present, chemotherapy is still to be the preferred and most significant therapeutic strategy for cancer patients in clinical practice. Although Camptothecin (CPT) has been discovered for over half century, a series of CPT derivatives such as Topotecan (TPT) and irinotecan (CPT-11) have been approved and are still to be the first-line medicines for clinical application. Up to now, the topoisomerase 1 inhibitor continues to be a significant drug development research field. Based on previous study of the structure-activity relationship, we consider that the introduction of lipophilic group at C7 position can prolong the retention time and the hydroxyl esterification at C20 can eliminate the hydrogen bond interaction, stabilize the E-lactone form and promote the anti-cancer effect. In this study, we carried out an optimization at C7 and C20 positions to afford two CPT derivatives 3g and 3j. Firstly, we predicted the possibly binding sites of two compounds with topoisomerase 1 by molecular docking. Then we evaluated the anti-proliferation effect of the two novel derivatives and compared the IC50 with CPT-11. Furthermore, the induction of cell cycle arrest and apoptosis was explored through karyomorphology, flow cytometry (FCM) and Western blot analysis. At last, we evaluated the anti-cancer effect and detected the mechanism in colorectal cancer xenograft model. In brief, all the data showed that the novel CPT derivatives (3g and 3j) could inhibit colorectal cancer proliferation via induction of cell cycle arrest and apoptosis in vitro and in vivo. It suggested that the two agents may be a new potential therapeutic strategy in the future.

Full text of DOI:10.1016/j.ejps.2018.08.018

EuropeanJournalofPharmaceuticalSciences123(2018)546–559
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Two novel camptothecin derivatives inhibit colorectal cancer proliferation
via induction of cell cycle arrest and apoptosis in vitro and in vivo
Hongzhi Dua,1, Yue Huanga,1, Xiaoying Houa,1, Xingping Quana, Jingwei Jiangb, Xiaohui Weia,
Yang Liua, Hongyang Lic, Puhai Wangd, Meixiao Zhane, Xun Aif, Ligong Lue,⁎, Shengtao Yuanb,⁎
Li Suna,⁎
,
a
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China
Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing, China
Hospital for Skin Disease, Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, Nanjing, China
Jiangsu Institute of Materia Medica, Nanjing Tech University, Nanjing, China
b
c
d
e
Center of Intervention Radiology, Zhuhai Precision Medicine Center, Zhuhai People's Hospital of Jinan University, Zhuhai, China
f
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, USA
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article:
CPT-11 (PubChem CID: 46781988)
At present, chemotherapy is still to be the preferred and most significant therapeutic strategy for cancer patients
in clinical practice. Although Camptothecin (CPT) has been discovered for over half century, a series of CPT
derivatives such as Topotecan (TPT) and irinotecan (CPT-11) have been approved and are still to be the first-line
medicines for clinical application. Up to now, the topoisomerase 1 inhibitor continues to be a significant drug
development research field. Based on previous study of the structure-activity relationship, we consider that the
introduction of lipophilic group at C7 position can prolong the retention time and the hydroxyl esterification at
C20 can eliminate the hydrogen bond interaction, stabilize the E-lactone form and promote the anti-cancer
effect. In this study, we carried out an optimization at C7 and C20 positions to afford two CPT derivatives 3g and
3j. Firstly, we predicted the possibly binding sites of two compounds with topoisomerase 1 by molecular
docking. Then we evaluated the anti-proliferation effect of the two novel derivatives and compared the IC50
with CPT-11. Furthermore, the induction of cell cycle arrest and apoptosis was explored through kar-
yomorphology, flow cytometry (FCM) and Western blot analysis. At last, we evaluated the anti-cancer effect and
detected the mechanism in colorectal cancer xenograft model. In brief, all the data showed that the novel CPT
derivatives (3g and 3j) could inhibit colorectal cancer proliferation via induction of cell cycle arrest and
apoptosis in vitro and in vivo. It suggested that the two agents may be a new potential therapeutic strategy in the
future.
Keywords:
Camptothecin derivatives
Colorectal cancer
Cell cycle arrest
Apoptosis
Proliferation
1. Introduction
of action being characterized as inhibitor of DNA topoisomerase I
(Topoisomerase 1) enzyme was defined by Hsiang et al. (1985).
At present, chemotherapy is still the preferred and most significant
treatment for cancer patients in the clinical application (Casado et al.,
2017), while chemotherapy, surgery, radiotherapy, biotherapy and
other adjuvant therapy are common treatments for tumor therapy
(Deng et al., 2015). The development of antitumor drug has been last
for over half century, plentiful chemotherapy drugs are still the first-
line medicines including Camptothecin (CPT). CPT, a broad-spectrum
antitumor agent, was first isolated from Chinese tree Camptotheca
acuminata by Wall and Wani (1996). Afterwards in 1985, its mechanism
Topoisomerase I is a ubiquitous nuclear enzyme with key role in
DNA replication and transcription. It catalyzes the cleavage and re-
ligation reaction of a DNA single-strand, by breaking or covalently
binding to a DNA phosphodiester bond (Luo et al., 2016; Rialdi et al.,
2016). CPT covalently trap (inserted into/targets at) DNA-Topo1
covalent complex, forming a DNA-Topoisomerase 1-CPT ternary com-
plex that inhibit the rejoining of a DNA single-stand break, produces
collisions between the replication and transcription forks, leading to
DNA fork breakage and DNA damage (Chen et al., 2013). As a result,
⁎
Corresponding author.
E-mail addresses: luligong1969@126.com (L. Lu), yuanst@cpu.edu.cn (S. Yuan), sunli@cpu.edu.cn (L. Sun).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejps.2018.08.018
Received 4 April 2018; Received in revised form 13 July 2018; Accepted 13 August 2018
Availableonline14August2018
0928-0987/©2018ElsevierB.V.Allrightsreserved.

H. Du et al.
EuropeanJournalofPharmaceuticalSciences123(2018)546–559
induction of cell cycle arrest is the typical event caused by DNA da-
mage. However, the DNA damage will simultaneously active DNA da-
mage response (DDR), leading to DNA damage repair. The DDR may
rescue the DNA damage to maintain cell survival while only serious
DNA damage surpassed DDR, resulting in apoptosis or death (Shimizu,
2006; Tang et al., 2017).
Screening Tool. 9 conformers were generated for each ligand and the
conformers with the lowest binding energy were selected.
Protein–ligand interaction was visualized using Pymol version 1.7.4.5.
2.3. Cell culture
Over the past decades, researchers had synthesized a series of CPT
derivatives by modifying the parent molecule of CPT to enhance its
efficacy and decrease its toxicity (F.Z. Li et al., 2017; Verma and
Hansch, 2009). Among them, CPT derivatives Topotecan (TPT) and
irinotecan (CPT-11) have been approved for use by FDA in cancer
treatments, such as colorectal cancer, lung cancer, ovarian cancer,
chronic myeloid leukemia and so on (Verma and Hansch, 2009). Un-
fortunately, resistance to CPT derivatives is observed in clinic. Mean-
while, the low solubility and high toxicity of CPT derivatives limited
their treatment applications in clinic (Li et al., 2006; Lian et al., 2017).
Therefore, it is urgent to develop novel inhibitor of topoisomerase I to
satisfy with clinical application (Lian et al., 2017; Park et al., 2017).
Such as Simmitecan (Hu et al., 2013), a novel inhibitor of topoisome-
rase I is being under clinical trial with the hope to be a new drug in
China (http://www.chinadrugtrials.org.cn/).
Based on previous study of the structure-activity relationship
(Dallavalle et al., 2006; Dallavalle et al., 2004), it was reported that
introduction of lipophilic group at C7 position is good at improvement
of the CPT's liposolubility and results in prolonging the retention time
in vivo. Moreover, the hydroxyl and carbonyl groups in ring E would
form an intra-molecular hydrogen bond interaction which is not ben-
eficial for the activity of CPT (Bom et al., 1999). In vivo, CPT remains a
balance of E-lactone form and Carboxylic salt form. The decrease of the
E-lactone form can reduce the anti-cancer effect, while the increase of
the Carboxylic salt form can lead to the toxic and side effects (Mi and
Burke, 1994). Therefore, we consider that the introduction of lipophilic
group at C7 position can prolong the retention time and the hydroxyl
esterification at C20 can eliminate the hydrogen bond interaction,
stabilize the E-lactone form and promote the anti-cancer effect.
In this study, we carried out an optimization at C7 and C20 positions
to afford two CPT derivatives 3g and 3j. First, we predicted that both 3g
and 3j might be the inhibitors of Topoisomerase 1 by molecular
docking. Moreover, the anti-proliferative effect, the induction of cell
cycle arrest and apoptosis were fully revealed in vitro. At last, we
evaluated the anti-cancer effect and detected the mechanism in color-
ectal cancer xenograft model. Importantly, we have obtained the
granted patent of 3g and 3j (CN 102532151A). Therefore, the two novel
inhibitors of topoisomerase I may be a potential therapeutic strategy in
the future.
Human colorectal cancer cell lines HCT 116, HCT-15 and HT-29
were purchased from the Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. HCT 116,
HCT-15 and HT-29 cells were cultured in RPMI-1640 medium (Gibico,
USA) with 10% fetal bovine serum (FBS; HyClone, USA), 100 U/mL
penicillin and 100 U/mL streptomycin (Gibco, USA). The cells were
incubated in a humidified atmosphere with 5% CO2 at 37 °C.
2.4. Cell proliferation inhibition assay
Cells were harvested during the logarithmic growth phase, and
seeded into 96-well plates at a density of 1.5 × 103 cells/well. After
incubated for 24 h, cells were treated with serial concentrations of 3g or
3j for 72 h. Afterwards, 20 μL of MTT solution (5 mg/mL) was added to
each well and the cells were incubated in a 5% CO2 atmosphere at 37 °C
for another 4 h. Then the supernatant was discarded and the formazan
crystals were dissolved in 150 μL DMSO. Subsequently, the absorbance
was measured at 570 nm via Tecan Microplate reader (Durham, NC,
USA). The concentration that caused 50% inhibition of cell viabilities
(IC50) values was calculated by GraphPad Software. Triplicate experi-
ments were performed in parallel for each concentration, and the re-
sults are shown as the mean
SD. The inhibitory ratio was calculated
using the following formula: inhibitory ratio (%) = [1 − (mean absor-
bance of experiment group)/(mean absorbance of control
group)] × 100.
2.5. Colony formation assay
Human colorectal cancer cells were seeded into 6-well plates (500
cells/well). After incubated for 24 h, cells were treated with serial
concentrations of 3g or 3j for 10–15 days. The media with drugs were
refreshed every 5 days. Then, the media were discarded, cells were
fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal
violet for 10 min, and then washed with double distilled water to clean
any residual dye. The colony formations were photographed and
counted.
2.6. kFluor555 Click-iT EdU assay
EdU incorporation assay was used to assess cell proliferation.
5 × 104 cells per well were seeded on coverslips in 12-well plates. After
incubated for 24 h, cells were treated with different concentrations of
3g or 3j. EdU (5-ethynyl-2-deoxyuridine) incorporation assay was per-
formed as the kFluor555 Click-iT EdU kit (KeyGEN BioTECH, China),
EdU labeling and detection followed the manufacturer's protocol. After
treated for 24 h, cells were cultured for another 2 h in media supple-
mented with 10 μM EdU, fixed with 4% paraformaldehyde for 15 min
and then incubated in 0.5% Triton X-100 for 20 min at room tem-
perature. Next, the cells on coverslips were washed with 3% BSA in
PBS, and incubated in 1× Click-iT EdU staining mix for 30 min at room
temperature in the dark. Subsequently, the EdU-stained cells were
washed with 3% BSA in PBS and counterstained with Hoechst 33342 for
30 min. The stained cells were mounted in standard mounting media
and imaged by inverted fluorescence microscopy (Olympus IX-71;
Olympus, Japan).
2. Materials and methods
2.1. Chemicals and agents
Melting points were determined in X-4 Digital Microscope Melting
Point apparatus and are uncorrected. Column chromatography was
carried out on gel 100–200 mesh. 1H NMR spectra were recorded at
500 MHz with RUKER AVANED AV-500 instrument. ESI-MS were re-
corded on Waters Q-TOF Micro TM instrument.
2.2. Molecular docking
The crystal structure of DNA topoisomerase 1 in complex with
Topotecan was retrieved from RCBS protein data bank (PDB code:
1K4T). 3D structures of 3g and 3j were prepared and geometrically
optimized by Avogadro version 1.2.0. Standard Ligand Molecule pre-
paration such as addition of hydrogen atoms, bond lengths and bond
angle correction, co-crystallized water molecules removal and energy
minimization were performed before docking. Ligands were subjected
to molecular docking using AutoDock Vina in the PyRX 0.8 Virtual
2.7. Cell cycle analysis
The cell cycle distribution was measured by flow cytometry using
Propidium staining (PI) staining Kit (Vazyme, China). The synchronized
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EuropeanJournalofPharmaceuticalSciences123(2018)546–559
Fig. 1. The synthetic route and molecular docking of two novel camptothecin derivatives. (A). Synthetic route of 3g and 3j. (B). Docked structure and interactions of
3g binding to Topoisomerase 1. Left panel shows the residues of Topoisomerase 1 involved in the binding of 3g. (C) Docked structure and interactions of 3j binding to
Topoisomerase 1. Left panel shows the residues of Topoisomerase 1 involved in the binding of 3j.
2.9. Annexin V/PI staining
Table 1
Anticancer effects of 3g and 3j on different cancer cell lines.
Cell apoptosis was determined by flow cytometry using Annexin V-
FITC/PI double staining. The cells were harvested with 0.25% Trypsin
after exposed to various concentrations of 3g or 10 μM CPT-11 for 48 h,
washed with PBS and re-suspended in 500 μL binding buffer. Then the
cells were stained by adding to 5 μL Annexin V-FITC and 5 μL PI for
30 min at 37 °C in the dark. Finally, the cells were detected and ana-
lyzed using a Becton Dickinso FACSCalibur flow cytometer with Cell-
Quest software.
Tumor
Cancer cells
IC50 (μM)
3g
3j
CPT-11
Gastric cancer
Lung cancer
Colon cancer
SGC-7901
A549
HCT 116
1.15
5.13
0.67
0.03
0.14
0.09
5.88
7.96
1.21
1.02
1.36
0.20
8.42
9.92
2.34
1.48
4.53
0.33
human colorectal cancer cells were cultured for 24 h, and then treated
with various concentrations of 3g or 3j in medium supplemented with
normal serum, respectively. After incubated for 24 h, cells were har-
vested with 0.25% Trypsin-EDTA and fixed in 70% ethanol overnight at
4 °C. Subsequently, the cells were washed with PBS and re-suspended in
500 μL staining buffer. After that, 10 μL RNase A and 25 μL PI were
added to the cell suspension, cells were incubated for 30 min at 37 °C in
the dark. The cell cycle was determined using a Becton Dickinso
FACSCalibur flow cytometer. Data was analyzed using Cell-Quest soft-
ware.
2.10. Western blot analysis
After treated for 48 h, cells were washed with cold PBS and lysed in
lysis buffer (Cell Signaling Technology, USA) with complete phospha-
tase and protease inhibitor mixture (Thermo, USA). Lysates were cen-
trifuged at 12,000g for 20 min at 4 °C, and the supernatant was col-
lected. The protein concentration was measured by Bradford protein
assay via the Bio-Rad protein assay kits (Bio-Rad, USA). An equal
amount of protein was separated by 8%–12% SDS-PAGE gels and
transferred onto PVDF membrane (Millipore). The membranes were
blocked with 5% BSA in TBS buffer containing Tween-20 (TBST) for
1 h, and then incubated with specific primary antibodies diluted in 5%
BSA buffer overnight at 4 °C. After washed with TBST buffer for four
times every 5 min, the membranes were incubated with secondary an-
tibodies diluted in TBST buffer for 1 h at room temperature. The im-
munoblots were visualized by ECL reagents (Millipore) via Gel 2000
image analyzer (Bio-Rad Laboratories, USA).
2.8. Cell morphology assay
Human colorectal cancer cells were seeded on coverslips in 12-well
plates at a density of 5 × 104 cells/well. After treated with 3g or 3j for
48 h, cells were fixed with 4% paraformaldehyde for 15 min and in-
cubated in 0.5% Triton X-100 for 30 min at room temperature.
Subsequently, the cells nuclei were stained with Hochest 33342 (5 μg/
mL) for 30 min in the dark, then washed with PBS and the stained cell
nuclei were visualized using inverted fluorescence microscopy
(Olympus IX-71; Olympus, Japan).
2.11. Colorectal cancer xenograft model
Female BALB/c athymic nude mice (4–5 weeks) with body weight
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EuropeanJournalofPharmaceuticalSciences123(2018)546–559
Fig. 2. Stronger growth inhibitory effects of 3g or 3j on human colon cancer HCT 116 and HCT-15 cells. (A, B) Effects of 3g or 3j on cell viability of different human
colon cancer HCT 116, HCT-15 and HT-29 cells. (C, D) 3g or 3j exhibited stronger inhibition on cell viability than CPT-11 on human colon cancer HCT 116 (C) and
HCT-15 (D) cells. Cells were seeded into 96-well plates, after 24 h of incubation, cells were treated with various concentrations of 3g, 3j or CPT-11 for 72 h, and cell
viability were detected by MTT assay. Data shown are the mean
SD of three independent experiments.
from 18 to 20 g were purchased from the Model Animal Research
Center of Nanjing University. All animals were housed under standard
conditions and cared for according to protocols approved by the
Experimental Animal.
considered statistically significant; **, value of P < 0.01 and ***, value
of P < 0.001 was considered very statistically significant.
3. Results
Care Commission in China Pharmaceutical University. As previously
reported (Du et al., 2017), human colorectal cancer HCT116 cells
(5 × 106) injected into the subcutaneous tissue of armpit, then the
tumor resected and cut into pieces (about 1 mm3) after the tumor vo-
lume got to 400–500 mm3. Subsequently, the pieces of tumor tissue
were transplanted subcutaneously into each nude mice. When the
average tumor volume (TV) got to about 100 mm3, the mice were
randomly divided into five groups with six individuals per group. Based
on the dose converted from irinotecan (CPT-11) in clinical practice and
published paper about camptothecin derivatives (Casado et al., 2017;
Kuramochi et al., 2017), the therapeutic dose for mice is 8 mg/kg. The
treatment groups were respectively intraperitoneally injected CPT-11,
3g and 3j with the same dose (8 mg/kg), while the negative group was
given an equal amount of vehicle. After 21 days, the final tumor volume
was measured, and we got the relative tumor volume (RTV). TV and
RTV were calculated by the following formula: TV (mm3) = A/2 × B2,
where A represents the longest diameter of tumor, and B represents the
shortest diameter. RTV = V21/V0, V21 represents the TV of day 21, and
V0 represents the TV of day 0.
3.1. Chemical synthesis and molecular docking of 3g and 3j
3.1.1. Preparation of 7-Ethylcamptothecin-20(S)-O-m-nitrobenzoate (3g)
7-Et-CPT (0.75 g, 2 mmol), EDCI (1.53g, 8 mmol) and DMAP
(1.47 g, 12 mmol) were added to a solution of m-nitrobenzoic acid
(1.00 g, 6 mmol) in 100 mL of CH2Cl2 with stirring at 2 °C for 15 min.
The mixture was stirred at room temperature for 2 h and was poured
onto 100 ml ice water. The obtained organic layer was washed with
dilute hydrochloric acid and saturated salt water in turn, and con-
centrated. The crude product was chromatographically separated with
CH2Cl2-THF as eluent. The pure product 3g (0.81 g) was obtained as
almost white powder, yield 77.1%, purity 98.97% (HPLC), mp
272–274 °C. 1H NMR (CDCl3, 500 MHz) δ: 1.11 (t, J = 7.5 Hz, 3H, 19-
H), 1.40 (t, J = 7.8 Hz, 3H, 30-H), 2.37–2.49 (m, 2H, 18-H), 3.20 (q,
2H, 29-H), 5.25 (d, J = 18.5 Hz, 1H, 5-H), 5.29 (d, J = 19.0 Hz, 1H, 5-
H), 5.49 (d, J = 17.0 Hz, 1H, 17-H), 5.79 (d, J = 17.0 Hz, 1H, 17-H),
7.25 (s, 1H, 14-H), 7.68 (m, 2H, 10, 27-H), 7.77 (t, J = 7.5 Hz, 1H, 11-
H), 8.13 (t, J = 9.8 Hz, 2H, 9,12-H), 8.40 (d, J = 7.5 Hz, 1H, 24-H),
8.46 (m, 1H, 26-H), 8.93 (s, 1H, 28-H). ESI-MS m/z: 526.2 (M + 1)+
.
2.12. Immunohistochemistry
3.1.2. Preparation of 7-Ethylcamptothecin-20(S)-O-p-triflouoromethylbenzoate
(3j)
The expression of P-cdk1 and Cleaved-PARP in tumor tissues was
detected as previously described (Du et al., 2018). Apoptotic cells in
tumor tissues were determined by TUNEL Biotin Labeling Apoptosis
Detection Kit (KeyGEN BioTECH, China), according to the manufac-
turer's protocol.
The compound 3j was prepared from 7-Et-CPT and p-tri-
flouoromethylbenzoic acid according to the method of compound 3g.
The pure product 3j (0.90 g) was obtained as a white powder, yield
82.0%, purity 99.60% (HPLC), mp 252–254 °C 1H NMR (CDCl3,
500 MHz) δ: 1.10 (t, J = 7.5 Hz, 3H, 19-H), 1.40 (t, J = 7.8 Hz, 3H, 30-
H), 2.34–2.47 (m, 2H, 18-H), 3.21 (q, 2H, 29-H), 5.24 (d, J = 18.5 Hz,
1H, 5-H), 5.29 (d, J = 19.0 Hz, 1H, 5-H), 5.47(d, J = 17.0 Hz, 1H, 17-
H), 5.78 (d, J = 17.0 Hz, 1H, 17-H), 7.26 (s, 1H, 14-H), 7.64 (t,
J = 7.5 Hz, 1H, 10-H), 7.78 (q, 3H, 11,25,27-H), 8.14 (q, 2H, 9,12-H),
2.13. Statistical analysis
Data were shown as the mean
SD of triplicate experiments.
Statistical analysis was performed by a Student's t-test or one-way
ANOVA using the GraphPad Prism 5.0 software. All comparisons are
made relative to untreated controls. *, value of P < 0.05 was
8,22 (d, J = 8.0 Hz, 2H, 24,28-H). ESI-MS m/z: 549.2 (M + 1)+
To further predict whether DNA topoisomerase 1 inhibition by 3g
.
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EuropeanJournalofPharmaceuticalSciences123(2018)546–559
(caption on next page)
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EuropeanJournalofPharmaceuticalSciences123(2018)546–559
Fig. 3. 3g or 3j inhibited cell proliferation on colon cancer HCT 116 and HCT-15 cells. (A–B) 3g or 3j inhibited cell proliferation on human colon cancer HCT 116 and
HCT-15 cells. Cells were treated with various concentrations of 3g, 3j or CPT-11 for 24 h respectively. Total cells and proliferation cells (green) were detected by
staining hoechst and keyFluor594 (EdU). (C–F) 3g or 3j inhibited the colony formation in human colon cancer HCT 116 and HCT-15 cells. Cells were seeded into 6-
well plates, after 24 h of incubation, cells were treated with various concentrations of 3g or 3j as shown in the figure. After 10 days of incubation, the colony
formations were photographed (C and E) and colony formation rates were analyzed (D and F). Data shown are the mean
SD of three independent experiments.
*P < 0.05, **P < 0.01, ***P < 0.001, significant difference compared with control. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
and 3j could be caused by a direct interaction of 3g/3j with DNA to-
poisomerase 1, we performed molecular docking of 3g and 3j against
DNA topoisomerase 1 inhibitor complex structure. 9 different docking
complexes were generated and binding affinity was determined based
on binding energy values. The predicted energy values for 3g and 3j
showed good binding affinity for DNA topoisomerase 1. Binding en-
ergies for all 3g poses were comprised between −11.9 and −10.3,
while binding energies for all 3j poses were comprised between −11.2
and −10.1. Fig. 1 displayed the protein ligand interaction as visualized
on Pymol software. We can observe from the Fig. 1B and C that 3g and
3j have several interacting residues. It showed that 3g interacted with
the residues ARG-364, HIS-367, ARG-488, ASN-491, LYS-493, ALA-499,
THR-501, VAL-502, GLY-503, LYS-532, ASP-533, while 3j interacted
with the residues GLY-363, ARG-364, GLN-421, LYS-493, THR-501,
LYS-532, ASP-533. Based on these observations we suggested that 3g
and 3j can bind to a similar pocket on DNA topoisomerase 1.
3.4. 3g and 3j inhibited cell proliferation in HCT 116 and HCT-15 cells
The anti-cancer effect had preliminarily been revealed in Fig. 2.
Then EdU assays and colony formation assay were performed to further
evaluate the effects of 3g and 3j on cell proliferation in HCT 116 and
HCT-15 cells. As presented in Fig. 3A, B and Supplement Fig. 1, the
proliferation of HCT 116 and HCT-15 cells were significantly inhibited
after treating with various concentrations of 3g, 3j or 10 μM CPT-11 for
24 h, respectively. The cell growth of HCT 116 and HCT-15 cells were
also detected by colony formation assay. As shown in Fig. 3C–F, ex-
posure of HCT 116 and HCT-15 cells to 3g or 3j induced a significant
reduction in colony formation, as compared with vehicle-treated cells.
Obviously, 3g and 3j could inhibit colorectal cancer proliferation in
vitro.
3.5. 3g and 3j induced different cell cycle arrest in HCT 116 and HCT-15
cells
3.2. 3g and 3j inhibited proliferation in different cancer cell lines
As novel CPT derivatives, 3g and 3j are DNA damage anti-tumor
drugs, which may induce cell cycle arrest. To determine whether 3g and
3j induced cell cycle arrest in HCT 116 and HCT-15 cells, cells were
treated with 3g or 3j for 24 h, then stained with PI and cell cycle dis-
tributions were analyzed via flow cytometric. After treated with 3g for
24 h, the number of HCT 116 or HCT-15 cells in G2/M phase was in-
creased significantly (Fig. 4A). Furthermore, the percentage of cells in
the G2/M phase was increased, as concentration-dependent (Fig. 4B).
These results indicated that 3g induced a G2/M cell cycle arrest in HCT
116 and HCT-15 cells in a concentration-dependent manner. Similarly,
synchronized cells were treated with 3j for 24 h. As shown in Fig. 4C
and D, the percentage of cells in G2/M phase increased at low con-
centrations (0.3–3 μM), the same with 3g. However, it's interesting to
find that 3j predominantly arrested cells during S phase at the con-
centration of 10 μM, which is different with 0.3–3 μM, and also different
with 3g in 10 μM. These results indicated that 3j could induce different
cell cycle arrests in colon cancer HCT 116 and HCT-15 cells at different
concentration. In brief, both 3g and 3j could induce cell cycle arrest in
HCT 116 and HCT-15 cells.
Firstly, we investigated the inhibitory effect of 3g or 3j on different
human cancer cells by MTT assay. As shown in Table 1, after treatment
with various concentrations of 3g for 72 h, the IC50 value of 3g on
gastric cancer SGC-7901 cells, lung cancer A549 cells and colon cancer
HCT 116 were 1.15
respectively. Similarity, the IC50 value of 3j on these cells was
5.88 0.20 μM respectively.
0.03 μM, 5.13
0.14 μM and 0.67
0.09 μM,
1.02 μM, 7.96
1.3 μM and 1.21
These results revealed that either 3g or 3j exhibited great cell toxicity in
different cancer cells, while human colon cancer HCT 116 cells were
more sensitive to both 3g and 3j.
3.3. 3g and 3j exhibited greater anti-cancer effect than CPT-11 in HCT 116
and HCT-15 cells
To further investigate the cytotoxicity of 3g or 3j on colon cancer
cells, the cytotoxicity was evaluated on three common human colon
cancer cell lines HCT 116, HCT-15 and HT-29 by MTT assay. As shown
in Fig. 2A and B, the cell viability was significantly inhibited in a
concentration-dependent manner after treating with series concentra-
tions of 3g or 3j for 72 h. The IC50 value of 3g were 0.67
0.67 0.15 μM and 2.18 1.13 μM in HCT 116, HCT-15 and HT-29
cells, respectively (Fig. 2A); that of 3j were 1.21 0.18 μM,
1.42 0.30 μM and 5.71 0.95 μM, respectively (Fig. 2B). These
results demonstrated that both 3g and 3j inhibited the cell viability on
human colon cancer cells. Moreover, HCT 116 and HCT-15 cells were
more sensitive to 3g and 3j. Therefore, HCT 116 and HCT-15 cell lines
were chosen for subsequent experiments.
To compare the anti-cancer effect of 3g and 3j with CPT-11 on HCT
116 and HCT-15 cell lines, MTT assay was performed after treatment.
According to the inhibition rate curve, we found that either 3g or 3j
exhibited more potent cytotoxicity than CPT-11 on both HCT 116 and
HCT-15 cells (Fig. 2C and D). The IC50 value of CPT-11 in HCT 116 and
0.09 μM,
3.6. 3g and 3j induced cell cycle arrest through inhibiting the formation of
cyclin-cdks complex
The cell cycle arrest induced by 3g and 3j has been confirmed, but
the mechanism still needs to be explored. In cell cycle progress (H.Y. Li
et al., 2017; Mukherjee et al., 2016), the cyclin-cdks complex plays a
key role. To explore the mechanism of the induction cell cycle arrest by
3g or 3j, we detected the protein expression involved in S or G2 cell
cycle in HCT 116 and HCT-15 cells. Since 3g predominantly induced
G2/M cell cycle arrest, we examined the key proteins of G2/M phase
cell cycle regulatory molecules. The results showed that 3g up-regu-
lated the expression of phosphorylated cdk1, which is the inactive state
of cdk1, however, it didn't affect the expression level of cyclin B1
(Fig. 5A). Similarly, we detected the expression level of cyclin B1, cy-
clin A2 and cdk1 that control G2/M and S phase cell cycle. As shown in
Fig. 5B, the expression of phosphorylated cdk1 was up-regulated, while
no significant changes in cyclin B1 and cyclin A2 expression. These
results indicated that both 3g and 3j induced cell cycle arrest by pro-
moting the inactive of cdk1, rather than inhibiting cyclins. Taken
HCT-15 cells were 2.34
0.33 μM and 4.42
0.56 μM, respectively.
In each cell lines, 3g exhibited 3- to 7-fold more potent than CPT-11,
with IC50 value consistently below 1 μM and 3j showed about 2- to 4-
fold potent compare to CPT-11. In brief, these results suggested that 3g
and 3j may be more potent anticancer agents than CPT-11 in human
colorectal cancer therapy.
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Fig. 4. 3g or 3j induced cell cycle arrest on human colon cancer HCT 116 and HCT-15 cells. (A–B) 3g induced G2/M cell cycle arrest in HCT 116 and HCT-15 cells.
(C–D) Different doses of 3j differentially triggered multiple cell cycle arrest in HCT 116 and HCT-15 colon cancer cells. Cells were exposed to various doses of 3g or 3j
for 24 h then cell cycle was analyzed by PI staining and flow cytometry. Cell cycle distribution was also analyzed. Data shown are the mean
SD of three
independent experiments.
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Fig. 6. 3g or 3j induced cell cycle arrests via ATM-Chk1/Chk2-Cdc25C pathway on human colon cancer HCT 116 and HCT-15 cells. Cells were exposed to various
doses of 3g (A) or 3j (B) for 24 h and the protein expression levels involved in the ATR/ATM-Chk1/Chk2-Cdc25C pathway were detected by western blot.
together, 3g induced G2/M cell cycle arrest in HCT 116 and HCT-15
cells, while 3j induced G2/M cell cycle arrest at low concentrations but
S cell cycle arrest at high concentrations. Nevertheless, both 3g and 3j
induced cell cycle arrest via the increase of inactive cdk1.
phosphorylated-Cdc25C similarly enhanced the rate of p-Cdc25C/
Cdc25C. As previously reported (Wei et al., 2013), the increase of p-
Cdc25C/Cdc25C may finally result in the activation of Cdk1. In brief,
these results suggested that both 3g and 3j induced cell cycle arrest via
the activation of ATM rather than ATR. They activated the downstream
Chk1/Chk2-Cdc25C pathway, leading to an increase of phosphorylated
cdk1 and inhibited the combination of cyclin-Cdk1 complex, which
regulated cell cycle progression.
3.7. 3g and 3j induced cell cycle arrest via ATM-Chk1/Chk2-Cdc25C
pathway in HCT 116 and HCT-15 cells
Next we want to explore the mechanism of the phosphorylation of
cdk1 affected by 3g or 3j. Considering that CPT derivatives are DNA
damage agents, we detected the upstream pathways of cdk1 (Chulu
et al., 2010). As shown in Fig. 6, after treatment with 3g or 3j for 24 h,
we found that there was no significant change in the expression of
phosphorylated-ATR, while the expression of phosphorylated ATM up-
regulated. Then, the phosphorylation level of the downstream kinases
Chk1 and Chk2 were also up-regulated. Furthermore, both 3g and 3j
could suppress the total Cdc25C, while the expression of phosphory-
lated-Cdc25C was almost unconverted in HCT 116 cells. As a result, the
rate of p-Cdc25C/Cdc25C was enhanced by both 3g and 3j. In HCT 115
cells, the total Cdc25C was almost insusceptible. But the increase of
3.8. 3g and 3j induced apoptosis in HCT 116 and HCT-15 cells
As CPT derivatives, 3g and 3j might be DNA damage agents. Our
above research has confirmed that 3g and 3j could induce cell cycle
arrest in HCT 116 and HCT-15 cells. However, the DNA damage will
active DNA damage response, leading to DNA damage repair, only
serious DNA damage cause cell death (Amin et al., 2018). The cell
nuclear morphological will change when apoptosis occurs, as well as
apoptotic proteins.
To explore whether 3g or 3j effect the nuclear morphology, cells
treated with 3g or 3j were stained with Hochest and imaged by
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Fig. 7. Effects of 3g or 3j on the nucleolus morphological on human colon cancer HCT 116 and HCT-15 cells. Cells were treated with various doses of 3g (A and B) or
3j (C and D) for 48 h and hours the nucleolus morphological were detected by Hochest staining and fluorescence microscopy.
fluorescence microscope. The results (Fig. 7) showed that drug-treated
cells presented morphological features of early apoptosis, such as bright
nuclear condensation and apoptotic bodies. To further confirm the
apoptosis in HCT 116 and HCT-15 cells, cells were stained with Annexin
V/PI and subjected to flow cytometric analysis. As shown in Fig. 8A and
B, after treated with 3g, the apoptosis rate increased from10.37% to
8.09%, 14.5 4%, 17.88% and 25.66% in HCT 116 cells, respectively.
Similarly, the percentage of apoptosis cells was raised from 9.72% to
13.89%, 10.90%, 54.37% and 69.14% in HCT-15 cells, respectively.
The results also showed that the apoptosis rates increased from 5.64%
to 10.80%, 22.33%, 10.67% and 15.66% in HCT 116 cells and from
3.21% to 39.47%, 43.13%, 56.81% and 58.20% in HCT-15 cells re-
spectively after treatment with 3j (Fig. 8C and D). Moreover, cells were
also exposed to CPT-11, and the apoptosis rate were 12.96% and
25.92% in HCT 116 and HCT-15 cells separately.
Generally, apoptosis in cells is accompanied with the changes in
apoptotic proteins, especially caspase families. The expression of
apoptotic proteins in cells were detected after treatment with 3g or 3j
for 48 h. The results showed that the protein level of PARP cleavage
(cleaved-PARP) was up-regulated, and the caspase family proteins such
as Caspase-3 and Caspase-9 were down-regulated in HCT 116 and HCT-
15 cells after treated with 3g or 3j (Fig. 9).
3.9. 3g and 3j inhibited the tumor growth via induction of cell cycle arrest
and apoptosis in vivo
The induction of cell cycle arrest and apoptosis has been confirmed
by a series of methods in vitro. Conclusively, we evaluated the anti-
cancer effect and detected the induction of cell cycle arrest and apop-
tosis in colorectal cancer xenograft model. As shown in Fig. 10A–D,
both 3g and 3j could inhibit tumor growth without influencing the body
weight of mice. The positive drug, CPT-11 exhibited significant anti-
cancer effect whereas it also sharply decreased the body weight sug-
gesting a serious toxic or side effects. Meanwhile, the TUNEL assay
showed that both 3g and 3j could induce apoptosis in vivo (Fig. 10E).
The associated proteins of cell cycle arrest (P-cdk) and apoptosis
(Cleaved-PARP) were induced by both 3g and 3j. In brief (Fig. 10F),
these data indicated that both 3g and 3j inhibited the tumor growth via
induction of cell cycle arrest and apoptosis in vivo.
4. Discussion
Although the first Camptothecin-like drug, CPT has been discovered
for over half century, a series of CPT derivatives such as TPT, CPT-11
and Belotecan have been approved and are still to be the first-line
medicines for clinical application (Tang et al., 2016; Verma and
Hansch, 2009). Up to now, the development of Topoisomerase 1 in-
hibitor continues to be a significant direction (Park et al., 2017; Wolff
et al., 2018). Such as a novel inhibitor of Topoisomerase 1, Simmitecan
(Hu et al., 2013) started the clinical trial in 2016 (http://www.
chinadrugtrials.org.cn/). Similarly, we have got two novel
All these data suggested that serious DNA damage surpassed DNA
damage repair, leading to apoptosis. Intriguingly, both the intrinsic and
extrinsic apoptotic pathways may be involved in 3g or 3j induced
apoptosis in HCT 116 and HCT-15 cells.
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Fig. 8. 3g or 3j induced apoptosis on human colon cancer HCT 116 and HCT-15 cells. Cells were treated with various doses of 3g, 3j or CPT-11 for 48 h and the
apoptosis rates were assessed by Annexin V/PI staining and analyzed by flow cytometry (A and C). The Y-axis shows the PI labeled population, and the X-axis shows
the FITC-labeled Annexin V-positive cells. The apoptosis rates were shown on the Histogram (B and D). Data shown are the mean
experiments.
SD of three independent
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Fig. 9. Effects of 3g or 3j on the expression of apoptosis-related proteins on human colon cancer HCT 116 and HCT-15 cells. Cells were treated with various doses of
3g (A) or 3j (B) for 48 h and the apoptosis-related proteins were analyzed by western blot.
camptothecin derivatives (3g and 3j) and evaluated the anti-cancer
effect in vitro and in vivo. Meanwhile, the induction of cell cycle arrest
and apoptosis was detected. Thereupon, we have obtained the granted
patent of 3g and 3j (CN 102532151A). The two inhibitors of Topoi-
somerase 1 may be a potential therapeutic strategy in the future.
3g and 3j are CPT derivatives with the potential for the inhibitor of
Topoisomerase 1, and the data from molecular docking fully confirmed
the possibility. Topoisomerase 1 is a ubiquitous nuclear enzyme with
key roles in DNA replication and transcription (Casado et al., 2017).
Then the inhibitor of Topoisomerase 1 could form a DNA-Topoisome-
rase 1-inhibitor ternary complex that inhibit the rejoining of a DNA
single-stand break, leading to DNA fork breakage and DNA damage
(Park et al., 2017). As a result, induction of cell cycle arrest is the ty-
pical event caused by DNA damage. Therefore, we detected the cell
cycle arrest in Figs. 4–6 after the evaluation of anti-proliferation in
Figs. 1–3. As expected, the cell cycle arrest was significantly induced by
both 3g and 3j. However, the DNA damage will simultaneously active
DDR, leading to DNA damage repair (Montenegro et al., 2016). Then
the DDR may rescue the DNA damage to maintain survival as shown in
Fig. 10F. Nevertheless, if serious DNA damage surpassed DDR, it will
result in apoptosis or death. In further experiment (Figs. 7–9), we
checked the induction of apoptosis by a series of analytical methods.
Taken together, 3g and 3j could inhibit colorectal cancer via induction
of cell cycle arrest and apoptosis by targeting topoisomerase 1.
The cell cycle arrest induced by 3g and 3j has been confirmed in Fig. 4,
but the regulatory mechanism is needed to be explored. As previously
reported (H.Y. Li et al., 2017; Mukherjee et al., 2016), the cyclin-cdks
complex plays a key role in cell cycle progress, such cyclinD-cdk4/6 reg-
ulates the G1 phase, cyclin E-cdk2 regulates phase G1 transform to phase
S, cycle A2-cdk1/cdk2 adjusts S phase progress, and the transform of G2 to
S is regulated by cyclin B1-cdk1. Moreover, the increase of cyclins or cdks
will promote the formation of cyclin-cdks complex, which generally pro-
mote the progress of cell cycle. In consideration of G2/M phase arrested by
3g and G2/M eS phase arrested by 3j (Fig. 4), we detected the markers (P-
cdk1 and Cyclin B1) (Zhao et al., 2016) of G2/M phase in 3g treatment
and the markers (P-cdk1, Cyclin B1, Cyclin A2) (Hung et al., 2017) of G2/
M eS phase in 3j treatment (Fig. 5). The data from the two inhibitors
showed that both 3g and 3j induced cell cycle arrest by increasing the
inactive of cdk1, while not decreasing cyclins. Therefore, we detected the
expression level of phosphorylated proteins involved in DDR, p-ATR, P-
ATM (sensors), p-Chk1, p-Chk2 (inductors), cdc25c and p-Cdc25c (effec-
tors) (Guo et al., 2015; Wang et al., 2015). In brief, all the results suggested
that both 3g and 3j induced cell cycle arrest via ATR/ATM-Chk1/Chk2-
Cdc25C pathway.
After 3g and 3j induced cell cycle arrest by targeting topoisomerase
1, DDR could rescue the damage leading to apoptosis. Whereupon we
confirmed the induction of apoptosis by analyzed the Annexin V/PI
staining and detected the expression of Caspase-3, Caspase-9 and
Cleaved PARP. According previous reported (Elmore, 2007), there are
two classical death pathways—an extrinsic (death-receptor) pathway
and an intrinsic (mitochondrial) pathway. Both the two classical death
pathways could induce apoptosis and regulate the expression of Cas-
pase-3, Caspase-9 and Cleaved PARP. Therefore, we will confirm the
key one from the two pathways by other detection methods.
As shown in the experiments in vitro (Figs. 2–9), our novel CPT
derivatives with lower IC50 but higher induction of cell cycle arrest and
apoptosis, exhibited anti-cancer effect markedly better than CPT-11.
However, 3g and 3j could inhibit the tumor growth but with lower
inhibition rate than CPT-11 at the same dose. In consideration of CPT-
11 with sharply decreased the body weight suggesting a serious toxic or
side effects, 3g and 3j might have better anti-tumor effect at higher
dose. Meanwhile, increasing reports (Siolas and Hannon, 2013; Tentler
et al., 2012) showed that patient-derived xenograft (PDX) model is the
perfect model to evaluate effects in vivo. We will further confirm the
antitumor effect in the PDX model to promote drug development. In
addition, we will also analyze the pharmacokinetics of the inhibitors to
comprehensively investigate the possibility for potential therapeutic
strategy in the future.
Taken together, we got two novel camptothecin derivatives with the
granted patent. Firstly, the possibility for the inhibitor of topoisomerase
1 was confirmed. Next, the anti-proliferation effect was evaluated and
compared. Meanwhile, the induction of cell cycle arrest and apoptosis
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Fig. 10. 3g or 3j inhibited the proliferation via induction of cell cycle arrest and apoptosis in vivo. (A). 3g or 3j had no effect on the weight of mice compared with the
control group. (B) The relative tumor volume was inhibited by 3g, 3j or CPT-11. (C). All the tumors were shown in every group. (D). The inhibition rate of every
group was shown. (E). The expression of P-Ckd1, Cleaved-PARP and Tunel was detected by IHC. (F). The schematic representation demonstrates the mechanism by
which 3g or 3j inhibits colon cancer via induction of cell cycle arrest and apoptosis.
was explored. At last, we evaluated the anti-cancer effect and detected
the mechanism in colorectal cancer xenograft model. Obviously, the
data suggested that the two inhibitors may be a potential therapeutic
strategy in the future.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejps.2018.08.018.
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