A new Schiff base coordinated copper(II) compound induces apoptosis and inhibits tumor growth in gastric cancer

Article abstract of DOI:10.1186/s12935-019-0801-6

Background: Gastric cancer, as a multifactorial disorders, shows cytological and architectural heterogeneity compared to other gastrointestinal cancers, making it therapeutically challenging. Cisplatin is generally used in clinic for gastric cancer treatment but with toxic side effects and develops resistance. Anti-tumor properties of copper and its coordinated compounds have been explored intensively in recent years. Methods: In this study, we synthesized a novel Schiff base copper coordinated compound (SBCCC) and examined its antitumor effects in two gastric cancer cell lines SGC-7901 and BGC-823 as well as a mouse model of gastric cancer. Results: The results show that SBCCC can significantly inhibit the proliferation of gastric cancer cells in a dose- and time-dependent manner. The IC50 of SBCCC in SGC-7901 and BGC-823 cells is 1 μM, which is much less than cisplatin's IC50. SBCCC induces apoptosis and causes cell cycle arrest at the G1 phase. SBCCC induces apoptosis via multiple pathways including inhibition of NF-κB, ROS production and autophagy. Conclusions: The synthesized SBCCC induced cancer cell death via inhibition of NF-κB, ROS production and autophagy. The multiple cell-killing mechanisms were important to overcome therapeutic failure because of multidrug-resistance of cancer cells. SBCCC, with a lower IC50 compared to cisplatin, could render it the potential to overcome the side-effect for clinical application.

Full text of DOI:10.1186/s12935-019-0801-6

et al. Cancer Cell Int
https://doi.org/10.1186/s12935-019-0801-6
(2019) 19:81
Xia
Cancer Cell International
Open Access
PRIMARY RESEARCH
A new Schif base coordinated copper(II)
compound induces apoptosis and inhibits
tumor growth in gastric cancer
Yan Xia1†, Xingkai Liu2†, Luping Zhang1, Jinzhu Zhang1, Chaoying Li3, Nan Zhang1*, Hong Xu1* and Yan Li4
Abstract
Background: Gastric cancer, as a multifactorial disorders, shows cytological and architectural heterogeneity com-
pared to other gastrointestinal cancers, making it therapeutically challenging. Cisplatin is generally used in clinic for
gastric cancer treatment but with toxic side efects and develops resistance. Anti-tumor properties of copper and its
coordinated compounds have been explored intensively in recent years.
Methods: In this study, we synthesized a novel Schif base copper coordinated compound (SBCCC) and examined its
antitumor efects in two gastric cancer cell lines SGC-7901 and BGC-823 as well as a mouse model of gastric cancer.
Results: The results show that SBCCC can signifcantly inhibit the proliferation of gastric cancer cells in a dose- and
time-dependent manner. The IC50 of SBCCC in SGC-7901 and BGC-823 cells is 1 μM, which is much less than cispl-
atin’s IC50. SBCCC induces apoptosis and causes cell cycle arrest at the G1 phase. SBCCC induces apoptosis via multi-
ple pathways including inhibition of NF-κB, ROS production and autophagy.
Conclusions: The synthesized SBCCC induced cancer cell death via inhibition of NF-κB, ROS production and
autophagy. The multiple cell-killing mechanisms were important to overcome therapeutic failure because of mul-
tidrug-resistance of cancer cells. SBCCC, with a lower IC50 compared to cisplatin, could render it the potential to
overcome the side-efect for clinical application.
Keywords: Schif base copper coordination compound, NF-κB, Autophagy, ROS
Schif bases derived from an amino and carbonyl com-
pound belong to an important class of ligands that coor-
dinated to metal ions. Schif base-metal compounds have
been reported to have promising antibacterial and anti-
tumor activities [6, 7], while the Schif base coordinated
with copper compounds shows the most prominent in
the class of molecules [8–13]. Schif base copper(II) com-
pounds efectively bind to DNA via the DNA grooves,
and the binding between the copper ion and DNA is
much stronger compared to other essential elements [14].
Te potential anticancer efects of Schif base copper
compounds are well reported in various cancer cell lines,
including oxidative DNA cleavage to kill cancer stem
cell-enriched cells (HMLER-shEcad) and bulk cancer
cells (HMLER) [15], inducing apoptosis in lung (A-549)
and breast (MDA-MB-231) human cancer cell lines [16],
and inducing necrotic cell death and autophagy in breast
Background
Gastric cancer is one of the most common gastrointes-
tinal tumors and the second leading cause of cancer-
related mortality [1, 2]. As a multifactorial disorders,
gastric cancer shows cytological and architectural het-
erogeneity compared to other gastrointestinal cancers,
making it therapeutically challenging. Subpopulation of
patients at early-stage of gastric cancers can be cured [3],
however most patients diagnose at advanced-stage and
show only partial response to standard chemo-agents and
targeting-molecules therapy [4, 5]. Terefore, fnding new
and efective anticancer drugs is of the highest priority.
*Correspondence: 13604334181@163.com; 514329822@qq.com
†Yan Xia and Xingkai Liu contributed equally to this work
1 Department of Gastroenterology, The First Hospital of Jilin University,
No. 71. Xinmin Street, Changchun 130021, Jilin, China
Full list of author information is available at the end of the article
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Xia et al. Cancer Cell Int
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Page 2 of 11
Synthesis of the SBCCC
cancer cells (MCF-7) [17]. Te anticancer efects of Schif
base copper compounds are also investigated in in vivo
studies. In a diethylnitrosamine-induced liver carcinoma
study, Schif base heterodinuclear copper(II)Mn(II) com-
plex is signifcantly decreased the incidence and the num-
ber of hepatic nodules in a dose-dependent manner via
regulating infammation response and apoptotic pathway
[18]. A nephrotoxicity study shows that, whereas cispl-
atin increases serum urea nitrogen and creatinine levels,
no increase in serum biochemical parameters is detected
in the animals treated with Schif base heterodinuclear
copper(II)Mn(II) complex [18]. While a major obstacle to
widespread use of cisplatin is the persistence of toxic side
efects and drug-resistance, Schif base coordinated cop-
per compounds may be a novel chemotherapeutic strat-
egy for antitumor therapy, with the potential to overcome
cisplatin’s shortcoming.
For synthesis of the SBCCC, 2 mmol of salicylic alde-
hyde (0.402 g) and 2 mmol of salicylic hydrazide were
dissolved in 80 mL of anhydrous ethanol and heated in
a water bath under refux for 7 h. Next, the sediment
was fltered at atmospheric pressure and recrystal-
lized twice to aford the Schif base ligands. Te cop-
per coordinated Schif base complexes were prepared
by dissolving the Schif base ligands (2 mmol) and cop-
per sulfate (1 mmol) in anhydrous ethanol (70 mL). Te
mixture was heated in a water bath under refux for 8 h,
fltered at atmospheric pressure, and then recrystal-
lized. Te synthesis equation for SBCCC was shown in
Fig. 1.
MTS assay
In this study, we synthesized a Schif base copper coor-
dination compound (SBCCC) and investigated its cell-
killing efect and the potential cell death pathway(s) in
two gastric cancer cell lines and a xenograft mouse model
of gastric cancer.
MTS assay was performed to determine cell prolifera-
tion and the tested samples were triplicated. In brief, cells
were seeded at 1×104 cells/well in a 96-well plate. When
80% confuence was reached, the cells were washed with
phosphate-bufered saline (PBS) and treated with SBCCC
at diferent concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4,
and 12.8 μM) diluted in culture medium. After 24 h treat-
ment, 10 μL MTS was added into each well. Optical den-
sity (OD) value was determined at 490 nm in each test
well using a microplate reader (BioTek, Winooski, VT,
USA). Cell growth inhibition rate was calculated accord-
ing to the equation: cell growth inhibition rate=(1−OD
of SBCCC treated cell/OD of control cell)×100%. Half
maximal inhibitory concentration (IC50) was calculated
by GraphPad Prism 7.
Methods
Cell lines and experimental reagents
Human gastric cancer cell lines, SGC 7901 (TCHu46)
and BGC823 (TCHu11), were purchased from the Cell
Bank of Shanghai Institute of Biochemistry and Cell Biol-
ogy, Chinese Academy of Sciences (Shanghai, China).
Te catalogue number of each cell line is indicated
in the bracket following its name. Dulbecco’s modi-
fed Eagle’s medium and RPML-1640 culture medium
were obtained from Termo Scientifc Co. (USA).
Te gastric cancer cells, SGC 7901 and BGC823, were
maintained in their respective medium in a 5% CO2
incubator at 37 °C. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium (MTS) agent was purchased from Promega
Corporation (USA). Te annexin V-FITC cell apoptosis
detection kit and the cell cycle kit were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Acridine orange/
ethidium bromide (AO/EB) staining solution, Hoechst
33258, N-acetyl cysteine (NAC), and 4,6-diamidino-
2-phenylindole, dihydrochloride (DAPI), Nω-nitro-l-
arginine methyl ester (L-NAME), ammonium pyrrolidine
dithiocarbamate (PDTC) were purchased from Sigma (St.
Louis, MO, USA). Antibodies for detection of nuclear
factor (NF)-κBp65, IκB, and p-IκB were obtained from
Cell Signaling Technology (Danvers, MA USA). Antibod-
ies for detection of LC3I, LC-3II, and Beclin-1 as well as
the secondary antibody were from Shanghai Biological
Engineering Company Limited (Shanghai, China).
Flow cytometry
Cell death was determined by fow cytometric analy-
sis (FACS Calibur, BD, USA) using an Annexin V-FITC
Apoptosis Detection kit according to the manufactur-
ers’ instructions. Cell growth arrest was detected by fow
cytometry using propidium iodide (PI) staining. In brief,
cells were seeded in a 6-well culture plate at a density of
1×105 cells per well. When 80% confuence was reached,
the cells were treated with SBCCC at a fnal concentra-
tion of 1 μM. Only culture medium was added as control
cells. After treatment for 12, 24, and 36 h, the cells were
collected and washed twice with PBS, and incubated with
100 μL of RNase A at 37 °C in a water bath for 30 min.
Te cells were then incubated with PI at 4 °C in the dark
for another 30 min. After completion of all staining steps,
the cells were washed with incubation bufer and resus-
pended in 500ul incubation bufer for fow cytomet-
ric analysis. FlowJo 7.6.1 software was used for the data
analysis.

Xia et al. Cancer Cell Int
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Fig. 1 The schematic diagram for synthesis of SBCCC
Dual AO/EB, Hoechst 33258 and DAPI staining
SBCCC, 50 μM PDTC, 1 μM SBCCC+50 μM PDTC
respectively for 24 h. After treatment, the cells were col-
lected and reseeded in 6-well plates at a cell density of
200 cells/well. Te cells were then cultured for 3 weeks
under saturated humidity conditions of 5% CO2 at 37 °C,
and the culture medium was changed every 3 days. Cell
colony formation was examined by hematoxylin staining
and the colony numbers were counted.
For the AO/EB staining and apoptosis analysis, cells were
seeded in 6-well culture plates at a density of 1×104 cells
per well in a 5% CO2 incubator at 37 °C. When 80% con-
fuence was reached, the cells were washed with PBS and
treated with 200 μL of the SBCCC at 1 μM for 36 h, while
only culture medium was added as the control cells. Te
treated cells were washed with PBS, and then stained
with respective solution, 50 μg/mL dual AO/EB solution,
10 μg/mL Hoechst 33258, and 5 μg/mL DAPI were added
to each well. Te positive staining and cell morphology
were examined and photographed using an inverted fuo-
rescence microscope (LX71, Olympus, Japan).
Western blot
Total protein was extracted from cells using lysis bufer
containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl,
5 mM EDTA, 1% Triton-X 100, 1% dithiothreitol, and
1% protease inhibitor cocktail. Te protein sample was
loaded at 40 μg and separated by 10% sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis and transferred
onto a polyvinylidene difuoride membrane (Millipore,
USA). Membranes were blocked with 5% (w/v) nonfat
dry milk dissolved in Tris-bufered saline plus Tween-20
(TBS-T; 0.1% Tween-20; pH 8.3) at room temperature
for 1 h, then incubated with respective primary antibod-
ies (NF-κB p65, 1:150; I κB, 1:1000; p-I κB, 1:1000; LC3I,
1:1000; LC-3II, 1:1000; Beclin-1, 1:1000; Bcl-2, 1:500;
Bax, 1:500; caspase-3, 1:1000; cleaved caspase-3, 1:1000;
cleaved poly(ADP-ribose) polymerase (PARP), 1:1000;
cytochrome C, 1:1000; iNOS, 1:1000; JNK, 1:1000;
p-JNK, 1:1000) at 4 °C overnight. After washing with
TBS-T, the membranes were incubated with polyclonal
anti-horseradish peroxidase-labeled sheep or rabbit IgG
(1:5000) secondary antibodies for 2 h at room tempera-
ture. Immunobands were visualized using an enhanced
chemiluminescence kit (GE Healthcare, USA), according
Monodansylcadaverine (MDC) staining
For the MDC staining, the cells were cultured as
described above. Te cells were treated with SBCCC at
a fnal concentration of 1 μM for 12, 24 and 36 h. After
treatment, the cells were washed with PBS stained with
MDC at 50 μM. Cells were incubated for 1 h in a 5%
CO2 incubator at 37 °C. Te positive staining and cell
morphology were examined and photographed using
an inverted fuorescence microscope (LX71, Olympus,
Japan).
Colony formation assay
For the colony formation assay, SGC-7901 cells and BGC-
823 cells were seeded in 6-well culture plates at a density
of 1×104 cells per well and were cultured in a 5% CO2
incubator at 37 °C. When 80% confuence was reached,
the cells were washed with PBS and treated with 1 μM

Xia et al. Cancer Cell Int
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Page 4 of 11
to the manufacturer’s instructions, and exposed to X-ray
Results
flm. β-Actin was used as a loading control.
SBCCC inhibits growth of gastric cancer cells
With SBCCC treatment, there were signifcantly
increases of inhibitory rates in the gastric cancer cells
when SBCCC dosing increased from 0.4 to 12.8 µm
(P<0.05). Both SGC-7901 cells and BGC-823 cells
showed a similar growth inhibition trend, in a dose-
dependent manner (Fig. 2a, left). IC50 of SGC-7901
cells and IC50 of BGC-823 cells were 1.101 μM and
0.9864 μM, respectively. When the logarithms of SBCCC
concentrations were plotted versus the inhibition rates,
IC50 showed as 1 μM for both gastric cancer cell lines
(Fig. 2a, right). Terefore, we chose 1 μM as an approxi-
mate IC50 to continue the further experiments.
Determination of reactive oxygen species (ROS)
For the measurement of ROS generation, cells were
seeded in 6-well culture plates at a density of 1×104
cells per well and were cultured in a 5% CO2 incubator at
37 °C. When 80% confuence was reached, the cells were
washed with PBS and treated with 1 μM SBCCC for 12,
24 and 36 h. For untreated controls, only culture medium
were added. After treatment, the cells were washed with
PBS and then added 1 mL of freshly prepared DCFH-
DA was added at a fnal concentration of 10 μM. Te
cells were incubated in the dark for 20 min at 37 °C. Te
positive staining and cell morphology were examined and
photographed using an inverted fuorescence microscope
(LX71, Olympus, Japan).
SBCCC induces cell death of gastric cancer cells
To investigate the cellular events, apoptosis and cell
growth arrest were determined by Flow cytometry. With
SBCCC treatment, the apoptotic rates (%) for SGC-7901
cells were 25.11 1.34 at 12 h, 32.53 2.54 at 24 h, and
53.03 5.67 at 36 h, respectively. Similarly, the apop-
totic rates (%) for BGC-823 cells were 28.87 2.34 at
12 h, 39.62 3.61 at 24 h, and 64.33 6.25% at 36 h,
respectively. Te apoptotic rates for both cell lines sig-
nifcantly increased in a time-dependent manner com-
pared to control cells (P<0.05) (Fig. 2b left). Te growth
arrest was further evaluated in both SGC-7901 cells and
BGC-823 cells with SBCCC treatment. For both cell
lines, the percentage of cells in the G1 phase continu-
ally increased during the SBCCC treatments for 12, 24,
and 36 h. Te increases of G1 phase cells were found
to be statistically signifcant compared to control cells
(P<0.05). In contrast, the cells in the S phase continually
decreased during the SBCCC treatments for 12, 24, and
36 h. Te decreases of S phase cells were found to be sta-
tistically signifcant compared to control cells (P<0.05).
No changes were observed in the percentage of cells in
the G2 phase from both cell lines treated with SBCCC
(Fig. 2b right). Based on these results, the growth arrest
was happened at G1 phase for both gastric cancer cells,
implying that the SBCCC blocked the cell progression to
S phase.
Xenograft mouse model with gastric cancer cell
Twelve male BALB/c nude mice were purchased from
the Guangdong Medical Laboratory Animal Center
(Foshan, China). Te animals were housed four per
cage, given indicated chow and tap water, and main-
tained at 22 °C and on a 12-h light/dark cycle. To estab-
lished tumor model, SGC-7901 cells were inoculated
subcutaneously at 106 cells/mouse into the right fank.
Tumor growth was monitored and tumor sizes were
determined by measuring the length and width using a
caliber. Tumor sizes were calculated using the formula:
tumor volume=length×width×width/2. When the
average tumor size reached at 5–6 mm3. Te 9 tumor-
burden BALB/c nude mice were injected with SBCCC at
40 µM/kg bodyweight, i.p., twice per day. Tree control
mice were treated with same volume saline. Te SBCCC
treated mice were sacrifced at week 1 and week 2 and the
harvested tumor were fxed in formalin. At week 3, the
last 3 SBCCC treated mice along with the saline treated
controls were sacrifced and the harvested tumor tissues
were fxed in formalin for a week, then the tumor size was
determined and tumor gross anatomy was photographed.
Tis animal experimental protocol was approved by the
Animal Care and Use Committee (College of Basic Medi-
cine, Jilin University).
SBCCC interferes with DNA structure in gastric cancer cells
Te copper(II) complexes facilitate the DNA cleavage,
and exhibited substantial cytotoxic activity against can-
cer cells [19]. Dual AO/EB, Hoechst 33258 and DAPI
staining were performed in both SGC-7901 and BGC-
823 cells treated with SBCCC. AO, a cell-permeant dye,
emitted green fuorescence when bounded to dsDNA
but emitted red fuorescence when bounded to ssDNA.
Tis unique characteristic of AO staining allowed us to
Statistical analysis
Te experimental data obtained in this study were statis-
tically analyzed using Microsoft Excel 2007 and Graph-
Pad Prism7. Te results were statistically analyzed by
one-way analysis of variance, and multiple diferences
were analyzed by Duncan’s test. Te results are presented
as mean standard deviation (SD), and P<0.05 was con-
sidered statistically signifcance.

Xia et al. Cancer Cell Int
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Fig. 2 SBCCC-induced cell death and growth arrest in SGC-7901 and BGC-823 cells. a Left, MTS cell proliferation assay (*P <0.05); right, IC50 of
SBCCC, calculation based on logarithms of SBCCC concentrations versus the inhibition rates. b Left, fow cytometry analysis for apoptosis (*P <0.05).
b Right, fow cytometry analysis for cell growth cycle (*P <0.05 for G1 phase; #P <0.05 for S phase)

Xia et al. Cancer Cell Int
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evaluate the cell-DNA structure afected by SBCCC. EB binding of Hoechst 33258 dye and DNA shows strong
only entered cells with damaged membranes and emit- blue compared to normal chromosome cells. DAPI dye
ting orange-red fuorescence when bound to concen- is not completely permeability. Te ability of perme-
trated DNA fragments. Terefore, dual AO/EB staining ability for DAPI is improved in apoptotic cells, in which
allowed us to detect early apoptotic cells, late apoptotic high blue fuorescence is stained in condensed chro-
cells, and dead cells. Te result indicated that gastric mosome and cracking DNA, shown abnormal margin
cancer cells with 1 μM SBCCC treatment showed grad- of nucleus. When SGC-7901 and BGC-823 cells were
ually orange color for 12 h. Te numbers of orange- treated with 1 μM SBCCC for 36 h, strong blue fuores-
stained cells were increased at 24 h, and a portion of cence of Hoechst 33258 and DAPI was found. In DAPI
the SBCCC treated cells turned to red and orange-red staining with high magnifcation, pyknosis and abnor-
at 36 h (Fig. 3a). Both Hoechst 33358 and DAPI were mal margin of nuclei was identifed (Fig. 3a). Taken
minor groove-bound and UV-excited. In chromosome together, SBCCC interfering with DNA structure could
condensation or DNA cracking changes, increased be an important mechanism for its cell-killing efect.
Fig. 3 SBCCC-induced apoptosis and autophagy associated cell death in vitro and tumor growth inhibition by SBCCC in vivo. a Dual AO/EB staining
(×200); Hoechst 33258 (×100) and DAPI staining (×400); b MDC staining (×200), h: hour, c left, gross anatomy of the tumors harvested from each
group of the xenograft mouse model. Right, the tumor volume from control mice and the mice with SBCCC treatment. h: hour; UT: untreated;
*P <0.05

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SBCCC promotes autophagy in gastric cancer cells
signaling because of less anti-apoptosis products being
produced. However, the cell colony formation number
in the cells with PDTC treatment was not as less as that
in the cells with SBCCC treatment (Fig. 4c), indicating
that other pathways might contribute to the cell death
by SBCCC, in addition to its NF-κB inhibitory efect.
Previous report showed that the copper(II) complexes
induced apoptosis could be dependent on ROS genera-
tion [24]. Terefore, we further examined the generation
of ROS in the gastric cancer cells with SBCCC treat-
ment. As presented in Fig. 4d, ROS generation in SGC-
7901 and BCG-823 cells was signifcantly increased in a
time-dependent manner. SBCCC treatment signifcantly
increased the expressions of iNOS and phosphorylated
JNK and the increased expressions were inhibited by
two inhibitors, N-acetyl-l-cysteine (NAC, an inhibitor
of ROS) and L-NAME (an inhibitor of NOS) but not the
NF-κB inhibitor PDTC.
Monodansylcadaverine (MDC) was reported as a spe-
cifc in vivo marker for autophagic vacuoles [20]. Tere-
fore, MDC staining was further performed to detect the
autophagic vacuoles in both of SGC-7901 and BGC-823
cells challenged by 1 μM SBCCC. Te result indicated
that the autofuorescent MDC (green fuorescence) grad-
ually increased in the two cell lines, in a time-dependent
manner. Computer-image quantifcation for the MDC
green fuorescence indicated that there was signifcantly
increases of the fuorescence intensity of MDC in cancer
cells with SBCCC treatment for 12, 24 and 36 h, com-
pared to untreated controls (P<0.05) (Fig. 3b).
SBCCC inhibits tumor growth in a xenograft mouse model
Based on the in vitro data, the anti-tumor efect of
SBCCC was further tested in vivo using a moue tumor
model established by SGC-7901 cells xenograft. Te
result indicated that SBCCC treated mice showed a
signifcantly decreased in tumor size compared to the
untreated mice. As shown in Fig. 3c, the tumor volume
in untreated group was 1463 mm3 87.79 versus that in
the treatment groups 783 mm3 66.91 (P<0.05) at week
134 mm3 18.82 (P<0.05) at week 2, and 67 mm3 6.81
(P<0.05) at week 3.
NF-κB was reported as a pleiotropic transcription
factor activated by low levels of ROS but inhibited by
antioxidants [25]. Further experiment was performed
to determine if the SBCCC induced apoptosis could be
ROS-dependent in addition to NF-κB signaling. Interest-
ingly, the ROS inhibitor (NAC) alone or in combination
with SBCCC did not signifcantly afect NF-κB signaling
(Fig. 5a). Although ROS could act as upstream signaling
of the NF-κB pathway, our results indicated that the cell-
killing efect of SBCCC were from both ROS-dependent
DNA cleavage and inhibition of NF-κB signaling. As
that SBCCC induced increases of autophagic vacuoles
(MDC staining), the important molecular targets of
autophagy were further evaluated. Te protein expres-
sions of Beclin-1 and LC-3II were signifcantly increased
in SGC-7901 and BGC-823 cells with SBCCC treatment
compared to the control cells (Fig. 5b). To further eluci-
date the autophagy signal linking to NF-κB or ROS path-
ways, NAC and PDTC were used in combination with the
SBCCC to treat SGC-7901 and BGC-823 cells. PDTC did
not afect SBCCC induced increases of Beclin-1 and LC-
3II (Fig. 5c), but NAC signifcantly attenuated the SBCCC
induced protein expressions of Beclin-1 and LC-3II
(Fig. 5d). Tese results suggested that SBCCC associated
autophagy was ROS-dependent but not via the NF-κB
signaling.
Potential signaling targets of cell death by SBCCC
As the SBCCC induced apoptosis had been demon-
strated by above experiments, we further investigated the
important components of apoptotic signaling in term of
apoptotic initiators and efectors to elucidate the poten-
tial anti-tumor mechanism(s). As expected, SBCCC
treatment signifcantly reduced the protein level of Bcl-2
but increased the protein levels of Bax, and reduced the
release of cytochrome C from mitochondria to cytosolc,
in a time-dependent manner in both cell lines. Moreo-
ver, leaved caspase-3, and cleaved PARP-1, a hallmark
of apoptosis [21], were found signifcantly increased in
the SBCCC treated gastric cancer cells compared to the
controls (Fig. 4a). Because activation of NF-κB signaling
was well established in gastric cancer [22], the critical
components in NF-κB signaling were further evaluated
in the two gastric cancer cell lines challenged by SBCCC.
Because pyrrolidine dithiocarbamate (PDTC) could pre-
vent degradation of I-κB and translocation of NF-κB
from the cytoplasm into the nucleus [23], it was used a
selective NF-κB inhibitor to investigate if the anti-cancer
efect of SBCCC was mediated through NF-κB inhibition.
Te results indicate that treatments with either 1 μM
SBCCC or 50 µM PDTC signifcantly inhibited (P<0.05)
I-κB phosphorylation and NF-κB nucleus translocation
(Fig. 4b), indicating that the SBCCC caused cell death,
at least in part, was mediated by inhibition of NF-κB
Discussion
Metal-based compounds were widely used in the treat-
ment of diseases [26]. Te discovery of cisplatin in 1960
was a milestone in the history of metal-based compounds
used in the treatment of cancers [27]. Cisplatin is highly
reactive and covalently binds to DNA to form DNA-cispl-
atin adducts, which in turn induce DNA damage, inhibit
cell replication, and induce apoptosis. Unfortunately, the

Xia et al. Cancer Cell Int
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Fig. 4 SBCCC induces cell apoptosis via inhibition of NF-κB signaling and ROS production. a Western blot analysis for protein levels of Bcl-2, Bax,
caspase-3, cleaved caspase-3, cleaved PARP-1, and cytochrome C (Cyto-c). h: hour; *P <0.05. b Western blot analysis for protein levels of p-IκB,
IκB and NF-κB. h: hour; *P <0.05. c Cell colony formation assay. h: hour; *P <0.05 vs control; #P <0.05 vs PDTC. d Left, ROS production detected by
DCFH-DA staining. Right, Western blot analysis for protein levels of iNOS, JNK, and p-JNK. h: hour
side-efects such as peripheral neuropathy and nephropathy cell proliferation, rendering the potential of copper-based
were concerned for clinical use, and the tumor cells could with less toxicity and avoiding resistance [31]. In this study,
develop resistance mechanisms to those platinum drugs by a new synthesized SBCCC was investigated in regards of its
repairing the DNA damage [28]. In past decade, there was a antitumor properties in two gastric cancer cell lines as well
shift in the research focus of metal-based antitumor drugs as a xenograft mouse model of gastric cancer. Te IC50 of
towards copper [29], an essential element of the human SBCCC for the two gastric cancer cells was 1 µM, which
body showing less toxicity during tumor treatment [30]. was much less than cisplatin IC50 which ranged from 2.5
Copper coordinated compounds were diferent from plati- to 50 µM in multiple human cancer cell lines [32]. Te
num in many aspects, including physiological distribution lower IC50 could render SBCCC with the potential of less
in the body, intracellular aggregation properties, inhibiting side-efect for clinical application. Te SBCCC induced cell

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Fig. 5 SBCCC induced autophagy in ROS-dependent signaling. a Western blot analysis for protein levels of p-IκB, IκB and NF-κB in SGC-7901 and
BCG-823 cells treated with SBCCC, NAC and combination of SBCCC and NAC. *P <0.05. b Western blot analysis for protein levels of LC3-I, LC-3II, and
Beclin-1 in SGC-7901 and BCG-823 cells treated with SBCCC for 12 h, 24 h and 36 h. h: hour; *P <0.05. c Western blot analysis for protein levels of
LC3-I, LC-3II, and Beclin-1 in SGC-7901 and BCG-823 cells treated with SBCCC, PDTC and combination of SBCCC and PDTC. *P <0.05. d Western blot
analysis for protein levels of LC3-I, LC-3II, and Beclin-1 in SGC-7901 and BCG-823 cells treated with SBCCC, NAC and combination of SBCCC and NAC.
*P <0.05. N Nucleus; C Cytoplasm
death was demonstrated by multiple techniques, including fragmentation, which were the signature features of apop-
fow cytometry, dual AO/EB staining, Hoechst 33258 stain- tosis reported by previous studies [33, 34].
ing, and DAPI staining. Te SBCCC-treated cells showed
Similar to the NF-κB inhibitor PDTC, SBCCC treat-
cytoplasmic shrinkage, membrane blebbing, and DNA ment signifcantly inhibited the NF-κB transactivation

Xia et al. Cancer Cell Int
(2019) 19:81
Page 10 of 11
Abbreviations
for the productions of apoptotic initiator and efector,
including Bcl-2, Bcl-xL, cleaved caspase-3, and cleaved
PARP-1. Our results agreed with previous reports in
which inhibition of NF-κB transactivation by Schif base-
derived metal complex [35, 36]. It was reported that
ROS induced apoptosis via destruction of mitochondrial
membranes, release of cytochrome C from mitochondria,
the downstream activation of the caspase system ensues
[37–39]. In our study, ROS was produced in the gastric
cancer cells with SBCCC treatment, indicating that ROS
production was a signifcant mechanism contributing
to cell death. Using inhibitors to block both ROS and
NF-κB, we demonstrated that the cell-killing efect of
SBCCC was attributed to not only mitochondrial apop-
tosis directly triggered by ROS-dependent DNA cleav-
age, but also amplifed apoptotic signaling via inhibition
of NF-κB transactivation. ROS-dependent autophagy
was reported as a novel strategy to kill multidrug-resist-
ant cancer cells [40, 41]. A previous study showed that
copper(II) complex induced cell death via ROS-medi-
ated autophagy [42]. Te ROS-dependent autophagy by
SBCCC was demonstrated in this study. When treated
with ROS inhibitor NAC, the SBCCC induced activa-
tion of autophagy was totally abolished. ROS acted as
upstream signaling for the autophagy pathway activation
with SBCCC treatment to induce the autophagy associ-
ated cell death. Te potential anti-tumor mechanisms of
SBCCC were addressed in this study including (1) inhi-
bition of NF-κB signaling; (2) ROS production; and (3)
autophagy. Our data suggested that SBCCC could be a
novel agent to overcome the multidrug-resistance of can-
cer cells, through various cell-killing mechanisms.
SBCCC: Schif base copper coordinated compound; AO/EB: acridine orange/
ethidium bromide; NAC: N-acetyl cysteine; L-NAME: N(ω)-nitro-l-arginine
methyl ester; DAPI: 4,6-diamidino-2-phenylindole, dihydrochloride; PDTC:
pyrrolidine dithiocarbamate; MDC: monodansylcadaverine; PARP: poly(ADP-
ribose) polymerase; ROS: reactive oxygen species.
Authors’ contributions
YX and XL worked for the most cell experiments, data analysis and manuscript
drafting. LZ and JZ worked partly for the cell experiment and data analysis.
JZ and CL worked on the animal models and data collection. NZ and HX
contributed to the experimental design, consultant for the clinical relevance
and manuscript writing. YL contributed to English checking and manuscript
revision. All authors read and approved the fnal manuscript.
Author details
1 Department of Gastroenterology, The First Hospital of Jilin University, No. 71.
Xinmin Street, Changchun 130021, Jilin, China. 2 Department of Hepatobiliary
and Pancreas Surgery, The First Hospital, Jilin University, Changchun, Jilin,
China. 3 College of Basic Medical Science, Jilin University, Changchun 130021,
China. 4 Division of Surgical Oncology, Department of Surgery, University
of Louisville, Louisville, KY 40202, USA.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Funding
This work was supported by the National Natural Science Founda-
tion of China (#81472662, #81401581).
Project of Education Department of Jilin Province (#2016453, #2016482).
Norman Bethune Program of Jilin University (#2012219, #2015208).
Project of Science and Technology Department of Jilin Prov-
ince (#20140414048GH).
Science and Technology Development Planning of Jilin Prov-
ince (#20130206063YY, #20150204006YY).
However, other mechanisms of SBCCC could also con-
tribute to cell death in addition to inhibition of NF-κB,
ROS production and autophagy. For example, a study
proposed that the net efect of Schif bases copper(II)
complex for cell death could be destruction of the struc-
tural integrity of cell membranes, regardless of apoptosis
[43]. Schif base copper complexes with ternary structure
could also serve as ligand to inhibit proteasome and to
induce apoptosis [44]. Nevertheless, further studies are
needed to investigate SBCCC in regards of other mecha-
nisms and potential clinical application.
Health Department of Jilin Province (#2015Q011).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional afliations.
Received: 24 January 2019 Accepted: 23 March 2019
Conclusion
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Te synthesized SBCCC induced cancer cell death via
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multiple cell-killing mechanisms were important to over-
come therapeutic failure because of multidrug-resistance
of cancer cells. SBCCC, with a lower IC50 compared to
cisplatin, could render it the potential to overcome the
side-efect for clinical application.
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