8-Cetylcoptisine, a new coptisine derivative, induces mitochondria-dependent apoptosis and G0/G1 cell cycle arrest in human A549?cells

Article abstract of DOI:10.1016/j.cbi.2018.11.005

Lung cancer is the worldwide leading cause of cancer-related death. Here, we described the synthesis and the anticancer activity of a novel coptisine derivative 8-cetylcoptisine (CCOP) on lung carcinoma in vitro and in vivo. CCOP inhibited the cell viability of A549, BGC-823, MDA-MB-231, HCT-116 and HepG2 cell lines. In A549 cells, CCOP induced apoptosis, G0/G1 cell cycle arrest and decreased mitochondrial membrane potential (MMP) in a dose-dependent manner. Western blot analysis showed that CCOP increased the expression of Bcl-2-associated X protein (Bax), cleaved caspase 3 and 9, while decreased B-cell lymphoma 2 (Bcl-2), cyclins D and E, cyclin dependent kinases (CDKs) 2, 4 and 6, along with the inactivation of the upstream phosphoinositide 3-kinase (Pi3k)/protein kinase B (Akt) signaling. Further in vivo studies showed that CCOP (10 mg/kg) significantly delayed tumor growth in A549 xenograft nude mice, which is stronger than that of coptisine (100 mg/kg). These data suggested that CCOP could be a candidate for lung cancer therapy.

Full text of DOI:10.1016/j.cbi.2018.11.005

Accepted Manuscript
8-Cetylcoptisine, a new coptisine derivative, induces mitochondria-dependent
apoptosis and G0/G1 cell cycle arrest in human A549ꢀcells
Bing Han, Pu Jiang, Heshan Xu, Wuyang Liu, Jian Zhang, Siqi Wu, Liangyu Liu,
Wenyu Ma, Xuegang Li, Xiaoli Ye
PII:
S0009-2797(18)31041-X
https://doi.org/10.1016/j.cbi.2018.11.005
CBI 8456
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 5 August 2018
Revised Date: 31 October 2018
Accepted Date: 12 November 2018
Please cite this article as: B. Han, P. Jiang, H. Xu, W. Liu, J. Zhang, S. Wu, L. Liu, W. Ma, X. Li, X. Ye,
8-Cetylcoptisine, a new coptisine derivative, induces mitochondria-dependent apoptosis and G0/G1 cell
cycle arrest in human A549ꢀcellsC,hemico-Biological Interactions (2018), doi: https://doi.org/10.1016/
j.cbi.2018.11.005.
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Graphical abstract

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8-Cetylcoptisine, a new coptisine derivative, induces mitochondria-dependent
apoptosis and G0/G1 cell cycle arrest in human A549 cells
Bing Hana,†, Pu Jianga,†, Heshan Xub, Wuyang Liua, Jian Zhangb, Siqi Wua, Liangyu
Liua, Wenyu Mab, Xuegang Lia,c,*, Xiaoli Yeb,c,**
a
Chongqing productivity promotion center for the modernization of Chinese
traditional medicine, School of Pharmaceutical Sciences, Southwest University,
Chongqing, 400716, China
b School of Life Sciences, Southwest University, Chongqing, 400715, China
c Chongqing Engineering Research Center for Pharmaceutical Process and Quality
Control, Chongqing, 400716, China
*Correspondence to: X. Li, School of Pharmaceutical Sciences, Southwest University,
Tiansheng Road 2, Beibei district, Chongqing, 400715, China. Tel./fax: +86
023-68250728.
**Correspondence to: X. Ye, School of Life Sciences, Southwest University,
Tiansheng Road 2, Beibei district, Chongqing, 400715, China. Tel./fax: +86
023-68250728.
E-mail addresses: Xuegangli@swu.edu.cn (X. Li), yexiaoli@swu.edu.cn (X. Ye).
†These authors contributed equally to this work.

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Abstract
Lung cancer is the worldwide leading cause of cancer-related death. Here, we
described the synthesis and the anticancer activity of a novel coptisine derivative
8-cetylcoptisine (CCOP) on lung carcinoma in vitro and in vivo. CCOP inhibited the
cell viability of A549, BGC-823, MDA-MB-231, HCT-116 and HepG2 cell lines. In
A549 cells, CCOP induced apoptosis, G0/G1 cell cycle arrest and decreased
mitochondrial membrane potential (MMP) in a dose-dependent manner. Western blot
analysis showed that CCOP increased the expression of Bcl-2-associated X protein
(Bax), cleaved caspase 3 and 9, while decreased B-cell lymphoma 2 (Bcl-2), cyclins
D and E, cyclin dependent kinases (CDKs) 2, 4 and 6, along with the inactivation of
the upstream phosphoinositide 3-kinase (Pi3k)/protein kinase B (Akt) signaling.
Further in vivo studies showed that CCOP (10 mg/kg) significantly delayed tumor
growth in A549 xenograft nude mice, which is stronger than that of coptisine (100
mg/kg). These data suggested that CCOP could be a candidate for lung cancer
therapy.
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Keywords: coptisine derivative; 8-cetylcoptisine; lung cancer; apoptosis; cell cycle
Abbreviations
Akt, protein kinase B; AO/EB, acridine orange/ethidium bromide; Bak, Bcl-2
homologous antagonist/killer; Bax, Bcl-2-associated X protein; Bcl-2, B-cell
lymphoma 2; CCOP, 8-cetylcoptisine; CDKs, cyclin dependent kinases; COP,
coptisine; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; DMEM, Dulbecco’s
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Modified Eagle’s Medium; MMP, mitochondrial membrane potential; MOMP,
mitochondrial outer membrane permeabilization; NSCLC, non-small-cell lung cancer;
PARP, poly ADP-ribose polymerase; PI, propidium iodide; Pi3k, phosphoinositide
3-kinase.
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1. Introduction
Lung cancer is the leading cause of cancer incidence and mortality worldwide.
According to International Agency for Research on Cancer, about 2.1 million new
cases and 1.8 million deaths are predicted in 2018 [1]. Non-small-cell lung cancer
(NSCLC) accounts for almost 80% of lung cancer cases. In recent years, the
combined use of chemotherapy and surgery has led to the improvement of patient’s
outcomes. However, the 5-year survival rate of lung cancer remains disappointing [2],
suggesting the need for the development of novel therapeutic strategies for lung
cancer treatment.
Apoptosis, also called programmed cell death, plays a critical role in the pathogenesis
of lung cancer. Numerous chemotherapeutic agents are reported to induce death in
cancer cells by apoptosis [3, 4]. It is well established that caspase proteins are key
modulators of apoptosis induction [5]. Upon activation, initiator caspases 8 and 9
cleave and activate downstream effector caspases 3, 6, and 7, which further execute
apoptosis by cleaving target proteins such as poly ADP-ribose polymerase (PARP) [6].
On the other hand, the B-cell lymphoma 2 (Bcl-2) family proteins such as Bcl-2 and
Bcl-2-associated X protein (Bax) regulate the mitochondrial outer membrane
permeabilization (MOMP) and activate mitochondrial-mediated apoptosis pathway
[7]. Dysregulation of phosphoinositide 3-kinase (Pi3k)/protein kinase B (Akt)
components promotes pro-apoptotic function of Bax and induces mitochondrial
membrane potential (MMP) reduction and reactive oxygen species production [8].
Besides, Pi3k/Akt pathway triggers a network positively regulates cell cycle transition
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which relies on the activation of a series of cyclins and cyclin dependent kinases
(CDKs) [9]. Inhibition function of CDKs 2, 4, 6 and cyclins D, E could induce G1/S
arrest [10], while down-regulation of CDK 1 and cyclin B1 induces G2/M arrest [11].
Thus, the inhibition of Pi3k/Akt signaling may be a target for human cancer treatment
[12].
Natural products are considered an important source of anticancer agents, for example,
paclitaxel from Taxus brevifolia, and camptothecin from Camptotheca acuminata [13,
14]. It has been reported that the antitumor activity of coptisine (COP) from Coptis
chinensis Franch (Ranunculaceae) against lung [15], colon [16, 17], liver [18], breast
[19] and bone cancer [20]. However, its usage is limited because of poor
bioavailability, e.g. the absolute bioavailability of COP is between 1.87% to 0.52%
[21]. Alkylation is frequently used for structural modification of natural products
which may enhance the bioavailability and activity [22]. Hu et al. reported that adding
an hexadecyl moiety to the C8 of berberine, an analogue of COP, increased the
maximal plasma concentration (Cmax) by 2.8-fold. The relative bioavailability of
berberine to the derivative was 7.7% [23]. In addition, Jiang et al. reported that adding
C4, C6, C8, C10 and C12 alkyl chain at C8 of COP inhibited proliferation and
enhanced the glucose-lowering effect in HepG2 cells [24]. Besides, 8-octylcoptisine
exhibited higher antimicrobial activity than COP, especially against gram positive
bacteria [25]. Based on this, we synthesized a new COP derivative 8-cetylcoptisine
(CCOP) and investigated the potential antitumor effect on lung cancer in vitro and in
vivo.
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2. Material and methods
2.1. Compound and reagents
CCOP was synthesized as shown in Scheme 1 [22]. Grignard reagents were prepared
from Mg ribbon (44 mmol) and hexadecyl bromate (40 mmol) in absolute THF (100
ml). To a mixture of Grignard reagents in absolute THF, the suspension of compound
1 (30 mmol) was added drop-wisely. The reaction mixture was stirred under N2 at 0ºC
for 1 h. After warming at room temperature, the solution was heated to reflux for 1 h.
Then the solvent was removed by evaporation, redissolved in ethyl acetate and
recrystallized in MeOH to obtain compound 2. To a stirred solution of compound 2
(10 mmol) in hot AcOH (100 ml), Br2 (10 mmol) was added drop-wisely and heated
under reflux for 1 h. After cooling down at room temperature, the precipitate was
filtered and washed with 10% Na2S2O5 solution, then with H2O and recrystallized in
MeOH to yield compound 3. Then, compound 3 was solvated in hot MeOH, reacted
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with AgCl and recrystallized in MeOH at -20ºC to obtain compound 4. H and C
NMR spectra were detected on a Bruker Ascend 400 spectrometer (Bruker Biospin,
Switzerland). Mass spectrum was recorded on LCMS-8030 (Shimadzu, Japan).
COP (> 95% by HPLC) was prepared from the rhizome of Coptis chinensis Franch
according to a previous method [26]. Antibodies against caspase 3, caspase 9, Bcl-2,
Bax, CDK 2, CDK 4, CDK 6, Cyclin D, Cyclin E and anti-rabbit IgG
HRP-conjugated secondary antibody were purchased from Proteintech Group Inc.,
China. Antibodies against Pi3k, Akt and β-actin were purchased from Bioss
Biotechnology Inc., China. Antibody against p-Akt was purchased from Cell
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Signaling Technology Inc., USA.
2.2. Cell culture
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Human A549, BGC-823, MDA-MB-231, HCT-116 and HepG2 cells were obtained
from Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1%
penicillin/streptomycin (Invitrogen, USA) and 10% fetal bovine serum (BBI life
sciences, China). The cells were grown at 37°C in a 95% humidified atmosphere with
5% CO2.
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2.3. Cell viability assessment
Cell viability was determined by MTT (Sigma-Aldrich, USA) assay [13]. Briefly,
cells were seeded in 96-well plate at 6 × 103 cell/well and incubated overnight. CCOP
was dissolved in DMSO solution and added to the wells with the final concentrations
of 0−2 µg/mL. After treated for 18 h, 20 µL MTT (0.5 mg/mL) was added to each
well and treated for 4 h at 37°C. The supernatant was replaced with DMSO and
measured at 490 nm. The cell viability was normalized to untreated cells.
2.4. Flow cytometry
Cells in log-phase growth were seeded at 2 × 105 cells/well. For apoptosis assay, cells
were harvested, washed and stained with Annexin-V/propidium iodide (PI) kit
(Sigma-Aldrich, USA) according to the manufacturer’s instruction. For cell cycle
assay, cells were fixed in 70% ethanol at 4°C overnight then stained with 40 µg/mL PI
and detected by flow cytometer (BD FacsVerse, USA). The results were analyzed by
Flow Jo software (Tree Star Inc., USA) [16].
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2.5. Acridine orange/ethidium bromide (AO/EB) staining
For AO/EB staining, cells were seeded at 4 × 104/well on 12-well culture plate and
treated with CCOP. After 24 h incubation, cells were harvest, resuspended and
incubated in 4 µg/mL AO/EB for 1 min in the dark. Cells were imaged under a
fluorescence microscope (Nikon Eclipse Ci, Japan). Red fluorescence represents
apoptosis cells while green fluorescence represents healthy cells. At least 500 cells
were counted and the apoptosis rate was calculated by the percentage of red
fluorescence cells in all cells [27].
2.6. MMP assay
The MMP was detected with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3),
Sigma-Aldrich, USA) as reported previously [28]. Lipophilic cation such as DiOC6(3)
was transported and concentrated within the mitochondrial matrix by the negative
membrane potential. After CCOP treatment, cells were incubated with 50 nM
DiOC6(3) for 20 min at 37°C, rinsed with PBS and subsequently detected by flow
cytometry (excitation 488 nm; emission 525 nm). The results were analyzed using
Flow Jo software. Cells with lower fluorescence indicate loss of MMP.
2.7. Western blot analysis
Total protein was extracted by RIPA buffer (BBI life sciences, China) containing
protease inhibitors, separated by 10−15% SDS-PAGE, and transferred into
polyvinylidene fluoride membrane. After blocked by 10% nonfat milk for 2 h,
antibodies against Pi3k, Akt, p-Akt, caspase 3, 9, Bcl-2, Bax, CDK 2, CDK 4, CDK 6,
Cyclin D, Cyclin E and β-actin were incubated at 4ºC overnight. Subsequently, the
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membranes were rinsed with TBST and incubated with anti-rabbit IgG
HRP-conjugated secondary antibody at room temperature for 2 h. The results were
visualized with ECL reagent (Bio-Rad, USA) and quantified using Image J software.
2.8. Immunohistochemistry studies
Paraffin-embedded tumor sections were dewaxed, rehydrated, antigen retrieval by 10
mM sodium citrate buffer (pH 6.0) at 98ºC for 15 min and endogenous peroxidase
blocked with 3% H2O2 for 15 min. The slides were blocked with serum and incubated
with Ki-67, cleaved caspase 3 and Bcl-2. After rinsed with PBS, the sections were
incubated with IgG HRP-conjugated antibodies for 1 h at room temperature and
further stained with 3, 3'-diaminobenzidine and haematoxylin solution. The images
were captured under a microscope and analyzed using Image Pro Plus software [29].
2.9. Animal experiments
BALB/c nude mice (4-weeks old) were purchased from Beijing Huafukang
Bioscience Co. Inc., China (permit number: SCXK-JING 2014-0004) and housed in
sterile filter-topped cages. All animal experiments were in accordance with the
Laboratory Animal Care and Use Committee of Southwest University (permit number:
SCXK-YU 2014-0002). After 7 days acclimation, A549 cells were subcutaneously
inoculated in the right foreleg (5 × 106 cells/mice, n = 5). The mice were treated with
5 mg/kg (CCOP-L), 10 mg/kg (CCOP-H) or 100 mg/kg (COP) by oral gavage for 25
days. Mice in normal control (NC) and tumor control (TC) groups were administered
with saline. Tumor volume was measured using a caliper every two days and
calculated as tumor length × tumor width2/2. At the time of sacrificing, tumors and
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other tissues were quickly dissected, weighed and stored at -80ºC.
2.10. Statistical analysis
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All values were expressed as mean ± SD. Differences between groups were analyzed
by one-way ANOVA using SPSS 20.0 software. Values at or below p < 0.05 were
considered as significant.
3. Results
3.1. Synthesis and identification
The synthetic process and chemical structure of CCOP is shown in Scheme 1 and Fig.
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1A. CCOP: C35H46NO4, yellow powder (> 95% by HPLC, Fig. 1B). C NMR (400
MHz, CD3OD) δ: 14.41 (C-16'), 23.71 (C-15'), 28.28 (C-5), 29.27 (C-3'), 30.25 (C-4'),
30.45 (C-5'), 30.61 (C-6'), 30.69 (C-7'), 30.74 (C8'-C13'), 30.77 (C-14'), 33.06 (C-2'),
33.35 (C-1'), 50.97 (C-6), 103.57 (-OCH2O-), 104.96 (-OCH2O-), 106.84(C-1),
108.68 (C-4), 115.28 (C-8a), 121.47 (C-11), 121.77 (C-13b), 122.91 (C-13), 124.24
(C-12), 131.86 (C-4a), 134.07 (C-12a), 139.49 (C-13a), 145.89 (C-9), 149.74 (C-8),
149.88 (C-10), 151.88 (C-2), 161.76 (C-3). 1H NMR (400 MHz, CD3OD) δ: 0.90 (t, J
= 6 Hz, 3 H, 16'-CH3), 1.29 [m, 24 H, -(CH2)12-], 1.46 (m, 2 H, 15'-CH2), 1.65 (m, 2
H, 2'-CH2-), 1.87 (m, 2 H, 6-CH2-), 3.21 (t, J = 6 Hz, 2 H, 1'-CH2-), 3.81 (t, J = 8 Hz,
2 H, 5-H), 6.09 (s, 2 H, -OCH2O-), 6.44 (s, 2 H, -OCH2O-), 6.94 (s, 1 H, 4-Ar-H),
7.58 (s, 1 H, 1-Ar-H), 7.84 (2 H, 11-Ar-H, 12-Ar-H), 8.55 (s, 1 H, 13-Ar-H)
(Supplementary Fig. S1, S2 and Table 1). Yield: 38.2%. Mass spectrum (ESI):
+
Calculated for C35H47NO4 ([M+H]+): 545.34, Found: 545.60 (Fig. 1C).
3.2. CCOP inhibited cancer cell viability
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The cell viability was determined by MTT assay. Treatment with CCOP resulted in a
dose- and time-dependent cytotoxicity in different cancer cells. The IC50 values were
2.12 µg/mL and 1.05 µg/mL against A549 cells at 24 h and 48 h, respectively. To
identify whether the cytotoxicity of CCOP was specific to A549 cells, other cancer
cell lines were used. Interestingly, similar results were observed in BGC-823,
MDA-MB-231, HCT-116 and HepG2 cells (Fig. 2A). It was further showed that
CCOP induced cell morphology shrinkage and detachment under a 2 µg/mL treatment
(Fig. 2B). Moreover, the clonogenic assay performed with a continuing treatment of
CCOP for 2 weeks showed markedly inhibitory effect on A549 cell growth (Fig. 2C).
These data validated that CCOP could inhibit proliferation of cancer cells.
3.3. CCOP induced apoptosis in A549 cells
To identify whether CCOP-induced cytotoxicity was due to apoptosis induction, we
employed Annexin-V/PI staining. The results showed that a significant apoptosis rate
was induced by CCOP. Especially, the percentage of apoptotic cells was increased to
29.58% (p < 0.01) in cells treated with 2 µg/mL CCOP (Fig. 3A). In Hoechst 33342
staining, CCOP induced apoptotic cells with condensed and fragmented nucleus in
A549 cells (Supplementary Fig. S3). Additionally, the AO/EB assay also revealed an
increase in the number of red-stained dying cells and decreased number of
green-stained healthy cells, confirmed that CCOP could induce apoptosis in A549
cells (Fig. 3B). Decreased MMP may be an early event in the process of apoptosis.
Therefore, we further investigated the effect of CCOP on MMP. As expected, CCOP
decreased the MMP in a dose-dependent manner. Compared with control, CCOP
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(0.25−2 µg/mL) increased the percentage of cells with low MMP to 19.33%, 22.70%,
26.03% (p < 0.05) and 34.37% (p < 0.01), respectively (Fig. 3C), suggesting
CCOP-induced apoptosis in A549 cells is associated with MMP disruption.
3.4. Effect of CCOP on apoptosis-related proteins
The mechanism of CCOP-induced apoptosis was explored by examining the
expression of apoptosis-related proteins. Interestingly, western blot analysis showed
that CCOP reduced the expression of anti-apoptotic Bcl-2 and increased the level of
pro-apoptotic Bax (Fig. 4A). This observation confirmed the MMP disruption in
CCOP incubated A549 cells. A modulation of Bcl-2 family proteins could activate
caspase-dependent apoptosis [30]. To investigate the participation of caspase 3 and 9
in the pro-apoptotic effect of CCOP in the A549 cells, we measured the expression of
these proteins after the cells exposed to the CCOP treatment. As shown in Fig. 4A,
CCOP incubation increased the expression of cleaved caspases 3 and 9 in a
dose-dependent manner. In addition, CCOP reduced the expression of upstream Pi3k
and p-Akt signaling (Fig. 4B). These observations suggested that CCOP induced
caspase-dependent apoptosis in A549 cells.
3.5. CCOP induced G0/G1 arrest in A549 cells
We also detected the effect of CCOP on cell cycle by flow cytometry. The results
showed that CCOP induced G0/G1 arrest in A549 cells. Compared with control,
CCOP increased the population of G0/G1 phase by 16.44%, 17.61% and 16.01% (p <
0.05) at the concentrations of 0.5−2 µg/mL, respectively, accompanied by a decrease
in S phase cells (p < 0.05). However, CCOP had no influence on G2/M phase (Fig.
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5A). During G0/G1 phase, CDKs and Cyclin D/E promote DNA replication and
initiate G1-to-S transition [10]. Therefore, we further examined the expression of
G0/G1 regulatory proteins. Relative to control, CCOP dose-dependently decreased the
expression of CDKs 2, 4 and 6, Cyclins D and E (Fig. 5B). The results supported that
CCOP could induce G0/G1 cell cycle arrest in A549 cells.
3.6. CCOP inhibited tumor growth in vivo
We further investigated whether CCOP could inhibit tumor growth in xenograft nude
mice. As shown in Table 2, the gain in body weight was significantly decreased
between TC and NC group (p < 0.01) at the end of the experiment, probably due to
the cancer cell inoculation. On the other hand, the body weight of CCOP treated mice
was not markedly different from those in TC and COP treated group (p > 0.05).
Similarly, the organ index among all groups showed no obvious variation, indicated
that CCOP is relatively safe in vivo. As shown in Fig. 6A and 6B, CCOP inhibited
tumor growth in A549 xenograft nude mice. After 25 days, treatment with CCOP
significantly decreased tumor weight by 19.0% (p < 0.05) and 58.2% (p < 0.01) in
CCOP-L (5 mg/kg) and CCOP-H (10 mg/kg) groups respectively, as compared to TC.
While high dose of COP (100 mg/kg) decreased only 7.9% of tumor weight compared
to TC (Fig. 6C). Markedly, western blot analysis revealed that CCOP increased the
expression of cleaved caspase 3, Bax and decreased Bcl-2 expression in tumor tissues
(Fig. 6D). Immunohistochemical analysis (Fig. 6E) showed that, when compared with
TC, CCOP-H decreased Ki-67 and Bcl-2 expression by 73.94% (p < 0.01) and 63.11%
(p < 0.05), while increasing cleaved caspase 3 expression by 184.45% (p < 0.01). This
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result supports the idea of apoptosis activation. However, the effect of COP was not
significant (p > 0.05). These data indicated that CCOP could effectively block the
progression of lung cancer in vivo and its anticancer effect was much better than that
of COP.
4. Discussion
Lung cancer is the most common malignancy worldwide, with a high risk of
metastasis and poor prognoses [31, 32], making the development of effective
therapies for lung cancer an urgency. Preliminary results showed that CCOP
significantly inhibited the proliferation of A549 and other cancer cell lines in vitro.
Further studies indicated that the inhibitory effect was due to the apoptosis induction
and G0/G1 cell cycle arrest. Rao et al. reported that COP inhibited A549 cell growth
at IC50 value of 18.09 µM and induced G2/M cell cycle arrest [15]. However, the IC50
value of CCOP was 1.49–2.56 µg/mL, about ten-fold lower than that of COP in other
cancer cell lines (IC50 > 27.13 µg/mL) [15, 16, 19, 33]. These results could be
explained by the introduction of a long alkyl chain, which may enhance the compound
lipophilicity.
Many cellular signals for life and death are regulated by Bcl-2 family proteins [34].
For example, Bcl-2 restrains MOMP and suppresses apoptosis [35]. Bax and Bcl-2
homologous antagonist/killer (Bak) undergo oligomerization to form a channel that
triggers the release of apoptotic factors [36]. In addition, a decrease in MMP causes
MOMP and mitochondria-dependent apoptotic pathway [34]. In this study, CCOP
treatment significantly decreased MMP and Bcl-2 expression. On the other hand, the
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treatment activated Bax, caspase 3 and 9 indicated that CCOP could induce
mitochondria dysfunction and activate apoptosis in A549 cells.
It has been reported that the abnormal activation of Pi3k/Akt pathway is associated
with tumorigenesis, apoptosis and metastasis [37], and inhibition of Akt
phosphorylation stimulates caspase-meditated apoptosis in malignancies including
lung cancer [38]. Our data revealed that CCOP markedly decreased the expression of
p-Akt and the upstream regulator Pi3k in a dose-dependent manner, suggesting that
CCOP might modulate the Pi3k/Akt signaling in A549 cells. P-Akt also regulates the
cell division cycle and initiates G1-to-S phase transition which is regulated by the
activation of cyclins (e.g. cyclins D and E) and CDKs (e.g. CDKs 2, 4 and 6) [10]. In
this study, CCOP reduced the expression of cyclins D and E, and consistently
decreased the expression of CDKs 2, 4 and 6. These data indicated that
CCOP-induced apoptosis in A549 cells was associated with the suppression of
Pi3k/Akt pathway.
Previous results from our lab showed that COP at a dose of 100 mg/kg significantly
inhibited colon tumor growth [16, 17]. However, it showed only a 7.9% inhibition in
A549 xenograft nude mice, probably due to the different types of cancer cells. In this
study, although the dose of CCOP (10 mg/kg) was only one-tenth of COP’s dose, it
showed 58.2% inhibition rate, almost 7-fold compared to COP. These results further
suggested that the anticancer activity of CCOP was more efficient than that of COP in
vivo. In the progression of cancer development, the metastasis causes major death [39].
However, because of the tumor model, there was not observed metastasis in this study.
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Nevertheless, in a mouse 4T1 spontaneous metastasis model, CCOP inhibited lung
metastasis in vivo (Supplementary Fig. S4). Together, these data suggest that CCOP
could block the progression of lung cancer.
5. Conclusion
In conclusion, we demonstrated that CCOP exerted anticancer activity by inducing
mitochondria-dependent apoptosis and G0/G1 cell cycle arrest in A549 cells (Fig. 7).
CCOP might be a potential candidate for lung cancer treatment. However, further
clinical trials are needed to support our viewpoint. Besides, our data also provided
evidences for the antitumor structural modification of COP.
Conflict of Interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by The National Key Research and Development Program
of China for Traditional Chinese Medicine Modernization (2017YFC1702600,
2017YFC1702605); Chongqing postgraduate research and research innovation project
(CYB18087); The National Key Research and Development Program of China for
Chinese Veterinarian Medicine Modernization and Green Cultivation Technology
(2017YFD0501504);
The
Special
of
Program
for
Common
and
Key Technological Innovations
Key Industries
in Chongqing
(cstc2016zdcy-ztzx10001); Industrial Technology System Program for Traditional
Chinese Herbs of Chongqing Municipal Agricultural Commission (2017[5]); the
County-University cooperation Innovation Funds of Southwest University (SZ201703,
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HZ201601); Construction Project of Key subjects of Traditional Chinese Medicine of
the State Administration of Traditional Chinese Medicine of the People’s Republic of
China.
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Table 1. 1H NMR and 13C NMR spectra of CCOP (400 MHz, CD3OD).
NO.
δH
δC
1
2
3
4
4a
5
7.58 (1 H, s)
106.84
151.88
161.76
108.68
131.86
28.28
6.94 (1 H, s)
3.81 (2 H, t, J = 8 Hz)
6
1.87 (2 H, m)
50.97
8
8a
9
10
11
12
12a
13
13a
13b
-OCH2O-
-OCH2O-
1'
2'
149.74
115.28
145.89
149.88
121.47
124.24
134.07
122.91
139.49
121.77
103.57
104.96
33.35
7.84 (1 H, s) (overlap)
7.84 (1 H, s) (overlap)
8.55 (1 H, s)
6.09 (2 H, s)
6.44 (2 H, s)
3.21 (2 H, t, J = 6 Hz)
1.65 (2 H, m)
33.06
3'
4'
5'
6'
1.29 (2 H, m) (overlap)
1.29 (2 H, m) (overlap)
1.29 (2 H, m) (overlap)
1.29 (2 H, m) (overlap)
1.29 (2 H, m) (overlap)
1.29 (12 H, m) (overlap)
1.29 (2 H, m) (overlap)
1.46 (2 H, m)
29.27
30.25
30.45
30.61
30.69
30.74 (overlap)
30.77
23.71
14.41
7'
8'-13'
14'
15'
16'
0.90 (3 H, t, J = 6 Hz)
Data were detected on a Bruker Ascend 400 spectrometer, chemical shifts (δ) were
expressed in ppm.

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Table 2. Effects of CCOP on body weight and organ index (%) in BALB/c nude
mice.
Groups
Body weight (g)
Heart (%)
Liver (%)
Spleen (%)
Lung (%)
Kidneys (%)
Beginning
End
NC
TC
14.90 ± 0.59
14.71 ± 0.60
14.91 ± 0.37
15.09 ± 0.57
14.74 ± 0.31
22.96 ± 1.22
19.64 ± 0.85**
19.12 ± 0.46
19.05 ± 1.56
18.64 ± 0.98
0.62 ± 0.12
0.64 ± 0.05
0.65 ± 0.08
0.64 ± 0.03
0.71 ± 0.10
6.05 ± 0.70
6.63 ± 0.26
6.48 ± 0.39
6.23 ± 0.60
6.36 ± 0.50
0.54 ± 0.08
0.53 ± 0.10
0.58 ± 0.07
0.60 ± 0.12
0.58 ± 0.10
0.70 ± 0.04
0.74 ± 0.04
0.70 ± 0.06
0.69 ± 0.04
0.73 ± 0.07
1.75 ± 0.11
1.80 ± 0.19
1.73 ± 0.08
1.81 ± 0.20
1.88 ± 0.16
COP
CCOP-L
CCOP-H
Organ index = weight of organ / body weight × 100%.
Data were presented as mean ± SD, n = 5. **p < 0.01 compared with NC group. NC,
normal control; TC, tumor control. COP, coptisine at a dosage of 100 mg/kg;
CCOP-L, CCOP at low dosage (5 mg/kg); CCOP-H, CCOP at high dosage (10
mg/kg).

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Figure captions
Scheme 1. Synthetic route of CCOP (R = -CH2-(CH2)14-CH3).
Fig. 1. Chemical structure and identification of CCOP. (A) Chemical structure. (B)
HPLC analysis. HPLC detection condition: Polaris 5 C18-A column (250 × 10.0 mm);
mobile phase, methanol : water (0.05% triethylamine, 0.1% phosphoric acid) = 87:13;
flow rate, 1 mL/min; injection volume, 10 µL; detection wavelength, 347 nm; column
+
temperature, 30ºC. (C) Mass spectra (ESI). Calculated for C35H47NO4 ([M+H]+):
545.34, Found: 545.60.
Fig. 2. Effects of CCOP on cancer cell viability. (A) Cells were treated with CCOP
(0−4 µg/mL) for 24 h or 48 h, and determined by MTT assay. Data were presented as
mean ± SD, n = 5. (B) Morphology of A549 cells treated with CCOP (0−2 µg/mL) for
24 h. Scale bar, 200 µm. (C) Colonies of A549 cells treated with CCOP (0−2 µg/mL)
for 2 weeks. The picture depicts the crystal violet-stained colonies and the cloning
efficiency compared with control. Data were presented as mean ± SD, n = 3. *p <
0.05 and **p < 0.01 compared with control.
Fig. 3. Effects of CCOP on apoptosis in A549 cells. (A) Cells were treated with
CCOP (0−2 µg/mL) for 24 h, the apoptotic cells were stained by Annexin-V/PI and
analyzed by flow cytometry. (B) Cells were resuspended in PBS and incubated with
AO/EB (4 µg/mL) for 1 min in darkness. The arrows indicate typical cell bodies
(healthy cell, green-stained; apoptotic cell, red-stained). Scale bar, 100 µm. Data were
presented as mean ± SD, n = 3. *p < 0.05 and **p < 0.01 compared with control. (C)
Cells were treated with CCOP (0−2 µg/mL) for 24 h, stained with DiOC6(3) and

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measured by flow cytometry. Cells with lower fluorescence indicate loss of MMP.
Data were presented as mean ± SD, n = 3. **p < 0.01 compared with control
Fig. 4. Effects of CCOP on apoptosis related proteins in A549 cells. Cells were
treated with CCOP (0−2 µg/mL) for 24 h. The expression of apoptosis related proteins
(A) Bcl-2, Bax, cleaved caspase 3 and 9 (B) Pi3k, Akt and p-Akt was determined by
western blot. Data were presented as mean ± SD, n = 3. *p < 0.05 and **p < 0.01
compared with control.
Fig. 5. Effects of CCOP on cell cycle in A549 cells. (A) Cells were treated with
CCOP (0−2 µg/mL) for 24 h, then, stained with PI and analyzed by flow cytometry.
Data were presented as mean ± SD, n = 3. *p < 0.05 and **p < 0.01 compared with
control. (B) The expression of cell cycle related proteins (CDK 2, CDK 4, CDK 6,
Cyclin D and Cyclin E) was determined by western blot. Data were presented as mean
± SD, n = 3. *p < 0.05 and **p < 0.01 compared with control.
Fig. 6. Effects of CCOP on tumor growth in vivo. BALB/c nude mice were
subcutaneously injected with A549 cells into the right armpit and orally administered
with CCOP or COP every day. (A) Image of excised tumors. (B) Tumor volume. (C)
Tumor weight. Data were presented as mean ± SD, n = 5. *p < 0.05 and **p < 0.01
compared with TC group. (D) Western blot and (E) immunohistochemistry analysis of
apoptosis related proteins in tumor tissue. Scale bars, 100 µm. Data were presented as
mean ± SD, n = 3. *p < 0.05 and **p < 0.01 compared with TC group. TC, tumor
control; COP, COP at a dosage of 100 mg/kg; CCOP-L, CCOP at low dosage (5
mg/kg); CCOP-H, CCOP at high dosage (10 mg/kg).

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Fig. 7. Proposed mechanism of CCOP in A549 lung cancer. CCOP inactivates
Pi3k/Akt signaling pathway, decreases mitochondrial membrane potential and
activates caspase-dependent apoptosis. Simultaneously, CCOP suppresses the
expression of cyclin D, cyclin E and their corresponding CDK 2, CDK 4, CDK 6,
resulting in G0/G1 cell cycle arrest.

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Figures
Scheme 1.