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O R I G I N A L R E S E A R C H
Oxymatrine Inhibits the Proliferation and Invasion
of Breast Cancer Cells via the PI3K Pathway This article was published in the following Dove Press journal:
Cancer Management and Research
Lin Guo 1
Tengfei Yang 2
1Department of Gastrointestinal and
Nutriology Surgery, Shengjing Hospital of
China Medical University, Shenyang,
LiaoNing 110004, People’s Republic of China; 2The Department of Social
Service, Shengjing Hospital of China
Medical University, Shenyang, LiaoNing
110004, People’s Republic of China
Purpose: Oxymatrine has been reported to possess anti-cancer activity, but its role in breast
cancer (BC) is weakly defined. We investigated the anti-cancer effects of oxymatrine in
human BC cells, and the underlying molecular mechanisms of these effects.
Methods: BC lines were treated with oxymatrine. The MTT assay was conducted to
evaluate cell viability. The cell cycle and apoptosis of BC cells were analyzed using flow
cytometry and Hoechst 33258 staining. Transwell™ assays were undertaken to measure the
migratory and invasive abilities of MCF-7 or MDA-MB-231 cells. Expression of phospha-
tidylinositol 3-kinase (PI3K), Akt, cyclin D1, cluster of differentiation (CD)K2, PARP,
Gsk3β, caspase-3, matrix metalloproteinase (MMP)2 and Bax at protein and RNA levels
was measured by Western blotting and quantitative real-time polymerase chain reaction.
Results: Oxymatrine inhibited the proliferation of BC cells in a time-dependent manner. It
induced apoptosis in a dose- and time-dependent way according to Annexin V and Hoechst
33258 staining. Oxymatrine could inhibit the invasion of BC cells as shown by the Transwell
assay. Oxymatrine inhibited expression of B-cell lymphoma-2 while increasing that of Bax as
well as increasing expression of caspase-3 and caspase-9. Addition of oxymatrine to BC cells
attenuated the PI3K/Akt signaling pathway cascade, as evidenced by dephosphorylation of
P13K and Akt.
Conclusion: Oxymatrine exerts its anti-tumor effects in BC cells by abolishing the PI3K
pathway. Oxymatrine may be a new compound for BC treatment.
Keywords: oxymatrine, breast cancer, PI3K/Akt, proliferation, apoptosis, invasion
Introduction Breast cancer (BC) is a major cause of cancer-related death for women. The
mortality arising from BC is attributed to metastatic spread of cancer cells to
vital organs, such as the liver, bone and lung.1 An estimated 2.1 million new
cases of BC worldwide were recorded during 2018.2
Breast tumors are characterized by their biologic complexity and heterogeneity.
Progression of BC cells is a multi-step process that involves the dysregulation of
the multiple genes that control cell survival. Oncology is focusing increasingly on
finding important signaling pathways and targeting the molecules that promote the
survival, proliferation and metastasis of tumor cells.
In addition to several types of surgical procedures, current treatment for BC
requires judiciously applied serial endocrine, chemotherapeutic and biologic therapies.
Surgery is the primary treatment for patients with early BC and improves long-term
survival, but it is not efficacious for individuals with advanced BC.3 Non-surgical
treatments for BC have been investigated. However, traditional non-surgical therapies
Correspondence: Lin Guo Shengjing Hospital of China Medical University, 36 Sanhao Street, Heping, Shenyang, LiaoNing 110004, People’s Republic of China Email [email protected]
Cancer Management and Research Dovepress open access to scientific and medical research
Open Access Full Text Article
submit your manuscript | www.dovepress.com Cancer Management and Research 2019:11 10499–10508 10499
http://doi.org/10.2147/CMAR.S221950
DovePress © 2019 Guo and Yang. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the
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are associated with significant toxicity. Therefore, the devel-
opment of novel treatments is required urgently.
Natural products play an important part in cancer treat-
ment. For example, a bitter-melon extract has been used for
the treatment of BC or head and neck cancer.4–6 Oxymatrine
(Figure 1A) is an alkaloid extracted from a traditional
Chinese herb. Oxymatrine has been reported to inhibit the
proliferation, cell cycle and angiogenesis of cancer cells,
promote the apoptosis of cancer cells, and reverse multi-
drug resistance in patients with cancer.7 Some studies have
reported the anti-cancer activity of oxymatrine in the pan-
creatic cancer cells,8 colon cancer cells,9 hepatoma cells,10
gastric cancer cells11 and osteosarcoma cells of humans.12
However, reports of the anti-cancer activity of oxymatrine
on human BC cells are lacking, a knowledge gap that we
sought to fill in the present study.
Materials and Methods Reagents Dulbecco’s modified Eagle’s medium (DMEM)-high glu-
cose was purchased from Gibco (Gaithersburg, MD, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) was purchased from Sigma–Aldrich (Saint
Louis, MO, USA). Rabbit polyclonal antibodies against gly-
ceraldehyde 3-phosphate dehydrogenase (GAPDH) and cas-
pase-3 and monoclonal antibodies against Bax and B-cell
lymphoma (Bcl)-2 (Abcam, Cambridge, UK) were used at
1:5000 dilution. Rabbit monoclonal antibody to phosphati-
dylinositol 3-kinase (PI3K), Akt, and Aktp-Thr308 (Santa
Cruz Biotechnology, Santa Cruz, CA, USA) was used at
1:1000 dilution. Rabbit monoclonal antibody to cyclin D1,
cluster of differentiation (CD)K2, Gsk3β and matrix metal- loproteinase (MMP)-2 (Proteintech, Chicago, IL, USA) was
used at 1:1000 dilution.
Cell Culture The BC lines MCF-7 and MDA-MB-231 and human
embryonic kidney (HEK)-293 cells (used as controls) were
purchased from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China). Cells were cultured in DMEM
containing 10% fetal bovine serum, 100 U/mL penicillin and
100 μg/mL streptomycin. All cell types were grown at 37°C in a humidified incubator in an atmosphere of 5% CO2.
MTT Assay MTT assays were conducted to evaluate cell viability, as
described previously. Briefly, MCF-7, MDA-MB-231 or
HEK-293 cells were seeded at 104/well in 96-well plates
and plated in 0.1 mL DMEM supplemented with indicated
concentrations of oxymatrine for 12, 24, 36 or 48 hrs. At
each time point, 10 μL of MTT solution (5 mg/mL) was added, followed by incubation for 4 hrs at 37°C. Then, the
medium was replaced by 150 μL of dimethyl sulfoxide (DMSO) solution, followed by incubation for another 10
mins to solubilize crystals. The absorbance was read at
490 nm using a microplate reader (Bio-Rad Laboratories,
Hercules, CA, USA).
Hoechst 33258 Staining MCF-7 cells were treated with the indicated concentrations of
oxymatrine for 24 hrs. After incubation, cells were fixed with
4% polyoxymethylene and washed thrice with phosphate-
buffered saline (PBS), followed by incubation with Hoechst
33258 (10 μg/mL) in the dark for another 5 mins and washed thrice with PBS. Cells were observed and photographed under
a fluorescence microscope.
Flow Cytometry MCF-7 cells were cultured with the indicated concentra-
tions of oxymatrine for 24 hrs and then apoptosis was
measured using the Annexin V-FITC Apoptosis
Detection kit. Cells were collected by trypsinization, cen-
trifuged at 1000 × g for 5 mins at room temperature,
resuspended in 195 μL of Annexin V-FITC Binding Buffer and mixed with 5 μL of Annexin V-FITC. Then, cells were stained in the dark for 10 mins. After that, cells
were centrifuged at 1000 × g for an additional 5 mins,
resuspended in 190 μL of Annexin V-FITC Binding Buffer and mixed with 10 μL of propidium iodide. Then, cells were kept in the dark and subjected to flow cytometry.
Experiments were repeated thrice, and the results were
analyzed using CellQuest™ (Becton Dickinson, Franklin
Lakes, NJ, USA).
Assays to Measure the Migration and
Invasion of Cells Transwell™ assays (Sigma–Aldrich) were undertaken to
measure the migratory and invasive abilities of MCF-7 and
MDA-MB-231 cells. The upper chambers were washed
with serum-free medium, with or without the addition of
20 μL of Matrigel™ (Corning Life Sciences, Corning, NY, USA) covering the surface of a polycarbonate membrane
for migration or invasion experiments. Cells (105) in
0.2 mL of serum-free DMEM treated with or without the
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Figure 1 Oxymatrine inhibits the proliferation of breast cancer cells. (A) Molecular structure of oxymatrine. (B) HEK-293, MCF-7 and MDA-MB-231 cells were cultured with the indicated concentrations of oxymatrine for the indicated times in 96-well plates. The MTTassay was carried out, and results are the mean ± SD of three experiments done in triplicate.
(C) MCF-7 and MDA-MB-231 cells were cultured with the indicated concentrations of oxymatrine for the indicated times in 96-well plates. The MTTassay was carried out to calculate the inhibition of cell proliferation by oxymatrine, and the results are the mean ± SD of three experiments done in triplicate. (D) HEK-293, MCF-7 and MDA-MB-231 cells were cultured with the indicated concentrations of oxymatrine for 24 hrs, and PI3K expression was measured by Western blotting. (E) HEK-293, MCF-7 and MDA-MB-231 cells were cultured with the indicated concentrations of oxymatrine for 24 hrs, and PI3Kexpression was measured by real-time RT-PCR. (F) MCF-7 cells were treated with DMSO alone or with the indicated concentrations of oxymatrine for 24 hrs, and PI3Kexpression was measured by Western blotting. (G) MCF-7 cells were treated with DMSO alone or the indicated concentrations of oxymatrine for 24 hrs, and PI3Kexpression was measured by real-time RT-PCR. Results represent the mean ± SD of three experiments done in triplicate. (H) MDA-MB-231 cells were treated with DMSO alone or the indicated concentrations of oxymatrine for 24 hrs, and PI3Kexpression was measured by Western blotting. (I) MDA-MB-231 cells were treated with DMSO alone or the indicated concentrations of oxymatrine for 24 hrs, and PI3Kexpression was measured by real-time RT-PCR. Results are the mean ± SD of three experiments done
in triplicate. **P < 0.01, compared with the control group.
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indicated doses of oxymatrine were plated in the upper
chamber of each chamber, whereas the lower surfaces
were filled with 0.5 mL of DMEM supplemented with
10% fetal bovine serum. After incubation for 24 hrs at
37°C, cells on the upper compartments were removed,
whereas the invaded cells in the lower parts were stained,
observed and counted under a high-power microscope.
Western Blotting Lysates of total cellular proteins were extracted using 100 μL of RIPA Lysis Buffer. Then, 60 μg of lysates resolved in gels after sodium dodecyl sulfate–polyacrylamide gel electrophor-
esis was transferred to nitrocellulose membranes through elec-
troblotting. Then, membranes were blocked with 5% blocking
solution for 1 hr. This was followed by incubation overnight at
4°C with primary antibodies obtained from Santa Cruz
Biotechnology (PI3K; 1:1000 dilution; sc-390916), Akt
(1:1000; sc-5298), Aktp-Tyr308 (1:1000; sc-271966), caspase-
9 (1:1000; sc-56076), Bax (1:1000; sc-7480), Bcl-2 (1:1000;
sc-7382), GAPDH (1:1000; sc-47724), caspase-3 (1:1000; sc-
7272), PARP (1:1000; sc-390771), cyclin D1 (1:1000;
sc-8396), CDK2 (1:1000; sc-6248), GSK-3β (1:1000; sc- 377213), MMP2 (1:1000; sc-13594) apart from PIP3 (1:800;
PAB22210), which was from Abnova (Taipei, Taiwan). Then,
membranes were washed thrice with TBSTand incubated with
horseradish peroxidase-conjugated secondary antibodies
(Sigma–Aldrich) for an additional hour. Immunoreactivity
was measured using Western Lighting Ultra (Pierce
Technology, Rockford, IL, USA).
Quantitative Real-Time Polymerase Chain
Reaction (PCR) Total cellular RNA was extracted by 1 mL of TRIzol™
Reagent according to the manufacturer (Invitrogen, Carlsbad,
CA, USA) protocols. Then, 1 mg of RNA was reverse-
transcribed to cDNA in a 20-μL system by an RT Reaction kit (Promega, Fitchburg, WI, USA). Real-time PCR was done
using an Mx3000P Real-Time PCR system (Applied
Biosystems, Foster City, CA, USA). The PCR program was:
40 cycles of 94°C for 15 s, 60°C for 10 s and 72°C for 20 s. All
procedures were repeated thrice. Gene expression was normal-
ized to that of β-actin. The mouse primer sequences (forward and reverse, respec-
tively) used were: PI3K, 5′- GGACCCGATGCGGTTAGAG-
3′ and 5′-ATCAAGTGGATGCCCCACAG-3′; Gsk3β, 5′-GT ATGGTCTGCTGGCTGTGT-3′ and 5′-GGGTCGGAAGAC
CTTAGTCC-3′; CDK2, 5′-GCCATTCTCATCGGGTCCTC
-3′ and 5′-ATTTGCAGCCCAGGAGGATT-3′; cyclin D1, 5′-
CCGAGGAGCTGCTGCAAATGGAGCT-3′ and 5′-TGAA
ATCGTGCGGGGTCATTGCGGC-3′; caspase-9, 5′-GGTGA
CCCCAGAATTGACCC-3′ and 5′-TCGACAACTTTGCTG
CTTGC-3′; Bcl-2, 5′-GGTGAACTGGGGGAGGATTG-3′
and 5′-GGCAGGCATGTTGACTTCAC-3′; Bax, 5′-AGCTG
AGCGAGTGTCTCAAG-3′ and 5′-GTCCAATGTCCAGCC
CATGA-3′; MMP9, 5′-CGCATCTGGGGCTTTAAACAT-3′
and 5′-TCAGCACAAACAGGTTGCAG-3′; β-actin, 5′-TCG TGCGTGACATTAAGGAG-3′ and 5′-
ATGCCAGGGTACATGGTGGT-3′.
Statistical Analyses Data are the mean ± standard deviation. Differences were
evaluated by one-way analysis of variance (ANOVA) with
least-square difference test. P < 0.05 was considered sig-
nificant. Statistical analyses were conducted using SPSS
v16.0 (IBM, Armonk, NY, USA).
Results Oxymatrine Repressed the Viability of BC
Cells The BC lines MCF-7 and MDA-MB-231 and HEK-293 cells
(control group) were treated by the indicated concentrations of
oxymatrine. At an established time, point, oxymatrine reduced
the viabilities of MCF-7 and MDA-MB-231 cells significantly
in a dose-dependent manner; stronger effects were observed in
MCF-7 cells, but weaker effects were seen in the control group
(Figure 1B). To explore the differences between effects on
these cells, we measured PI3K expression at RNA and protein
levels: higher PI3K expression was noted in MCF-7 cells than
in MDA-MB-231 cells, with lowest expression observed in
the control group (Figure 1D and E). Oxymatrine inhibited
PI3K expression in MCF-7 cells and MDA-MB-231 cells in
a dose-dependent manner (Figure 1E, F, H and I).
Oxymatrine Expedited the Apoptosis
of BC Cells Hoechst 33258 staining showed that oxymatrine accelerated
chromatin condensation in MCF-7 cells to induce nuclear
degradation (Figure 2A). Data from flow cytometry also
demonstrated that oxymatrine treatment generated more apop-
totic cells (7.63% and 1.36% at 30 μM and 10 μM of oxyma- trine, respectively) than that in the control group (Figure 2B).
Oxymatrine treatment decreased the level of Aktp-Thr308
protein, with little change in the total Akt level (Figure 2C
and D). Also, oxymatrine treatment increased the expression
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Figure 2 Oxymatrine induces the apoptosis of MCF-7 cells. (A) MCF-7 cells were pre-incubated with the indicated concentrations of oxymatrine for 24 hrs, and then cells were stained with Hoechst 33258 and observed with a fluorescence microscope. (B) MCF-7 cells were pre-incubated with the indicated concentrations of oxymatrine for 24 hrs, and cells were treated with ANNEXIN-V-FITC and analyzed by FACS analysis. The experiment was repeated thrice. (C) MCF-7 cells were treated with DMSO or the indicated concentrations of oxymatrine for 24 hrs, and expression of PI3K, AKT, Akt
p-Thr308 caspase-3, Bax and Bcl-2 was measured by Western blotting. (D) MCF-7 cells
were treated with DMSO or the indicated concentrations of oxymatrine for 24 hrs, and mRNA expression of caspase-3, Bax and Bcl-2 was measured by real-time RT-PCR.
Results are the mean ± SD of three experiments done in triplicate. **P < 0.01, compared with the control group.
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of caspase-3 and Bax significantly at protein and RNA levels,
whereas Bcl-2 expression was downregulated.
Oxymatrine Suppressed Proliferation
of BC Cells We examined the effects of oxymatrine on the cycle of MCF-7
cells by flow cytometry. Oxymatrine arrested a greater propor-
tion of cells in the G1 phase (67.25% and 63.24% at 30 μM and 10 μM of oxymatrine, respectively), and a smaller proportion in the S phase (17.69% and 20.11% at 30 μM and 10 μM of oxymatrine, respectively) than the control group (Figure 3A).
Cyclin D1, CDK2 and Gsk3β have central roles in regulating the G1 phase of the cell cycle, so we measured their expression
in cells treated at different doses of oxymatrine. Western blot-
ting showed that exposure to the indicated concentrations of
oxymatrine for 48 hrs decreased expression of cyclin D1,
CDK2 and Gsk3β markedly (Figure 3B). Real-time PCR showed that oxymatrine inhibited the expression of cyclin D1
in a dose-dependent manner (Figure 3C). These results sug-
gested that oxymatrine suppressed the proliferation of BC
cells.
Oxymatrine Inhibited the Migration and
Invasion of BC Cells Τranswell assays with or without Matrigel were undertaken to test the inhibitory effect of oxymatrine on the migration
Figure 3 Oxymatrine suppressed the proliferation of breast cancer cells. (A) MCF-7 cells were pre-incubated with or without oxymatrine for 24 hrs, and then cells were analyzed using a FACS Vantage flow cytometer with CellQuest™ acquisition and analysis software. (B) MCF-7 cells were treated with DMSO or the indicated concentrations of oxymatrine for 24 hrs, and expression of cyclin D1, CDK2 and Gsk3β was measured by Western blotting. (C) MCF-7 cells were treated with DMSO or the indicated concentrations of oxymatrine for 24 hrs, and mRNA expression of cyclin D1, CDK2 and Gsk3β was measured by real-time RT-PCR. Results are the mean ± SD. Experiments were repeated thrice. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
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Figure 4 Oxymatrine inhibited the migration and invasion of breast cancer cells. (A and B) After pre-incubation with oxymatrine, MCF-7 cells were detected by Transwell™ without or with Matrigel™. (C and D) After pre-incubation with oxymatrine, MDA-MB-231 cells were detected by Transwell without or with Matrigel. (E) MCF-7 cells were treated with DMSO or the indicated concentrations of oxymatrine for 24 hrs, and MMP9 expression was measured by Western blotting. (F) MCF-7 cells were treated with DMSO or the indicated concentration of oxymatrine for 24 hrs, and mRNA expression of MMP9 was measured by real-time RT-PCR. Results are the
mean ± SD. Experiments were repeated thrice. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
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and invasion of BC cells, respectively. Oxymatrine reduced
the migratory and invasive abilities of MCF-7 cells in
a concentration-dependent manner (Figure 4A and B),
whereas similar (but weaker) inhibitory effects were
observed in MDA-MB-231 cells (Figure 4C and D). We
demonstrated that MMP9 expression was reduced at protein
and RNA levels in MCF-7 cells (Figure 4E and F).
Discussion Oxymatrine is considered to be a novel anti-tumor agent in
different types of cancer cells because it can retard pro-
liferation and the cell cycle and induce apoptosis.
However, its effects on BC cells are not known.
Here, we demonstrated that oxymatrine repressed the
proliferation, migration and invasion of MCF-7 and MDA-
MB-231 cells efficaciously in a dose-dependent and time-
dependent manner. Also, we demonstrated that oxymatrine
could inhibit PI3K expression.
The PI3K/Akt signaling pathway has a pivotal role in
regulating the apoptosis, proliferation and motility of
cells.13,14 Also, PI3K exhibits higher expression in tumor
cells than that in normal cells, suggesting that PI3K is
involved in the functions of activated tumor cells, and that
suppression of its expression could be an important strategy
against cancer.15 Reports have shown that fangchinoline
(traditional Chinese herb with anti-tumor activity) markedly
inhibited proliferation of SGC7901 cells (human gastric
tumor line) if high expression of PI3K occurred, but had
weaker inhibitory effects on MKN45 cells if PI3K was
expressed at a low level.16 In our study, higher expression
of PI3K was observed in MCF-7 and MDA-MB-231 cells
than that in HEK-293 cells, data that are consistent with the
results showing oxymatrine to have more obvious inhibitory
effects on MCF-7 and MDA-MB-231 cells than in normal
cells. Furthermore, Western blotting showed that oxymatrine
significantly decreased PI3K expression in a dose-
independent manner. Taken together, oxymatrine exerted its
anti-tumor ability via inhibition of PI3K expression.
The proliferation and division of cells are regulated in the
cell cycle by complex machinery comprising cyclins and
cyclin-dependent protein kinases (CDKs).17 Regulation of
the G1 phase of the cell cycle has attracted attention as
a target for the study and therapy of BC.18 Cyclin D is
a downstream locus of the PI3K/Akt signaling pathway.
Cyclin D forms a complex with CDK2 (or other types of
CDKs) to promote G1-phase progression towards the
S phase.19 Consistent with those elaborations, fluorescence-
activated cell sorting (FACS) analysis in our study
demonstrated that oxymatrine arrested the cycle of MCF-7
cells at the G1 phase, along with the reduced expression of
cyclin D1, CDK2 and GSK3β in the oxymatrine-treated group,
suggesting that oxymatrine suppressed proliferation of BC
cells dramatically.
Frequently, the growth of tumor cells is due to an imbal-
ance between cell proliferation and apoptosis. Caspase-3 is
a crucial regulator involved in apoptosis because it enhances
chromatin condensation and nuclear decomposition.20
Moreover, that balance between Bax expression and Bcl-2
expression is a major factor determining the apoptotic fate of
cells,21,22 and it has been an efficacious therapeutic target
for BC.23,24 Thus, we measured the expression of these reg-
ulators by Western blotting: expression of caspase-3 and Bax
was downregulated after oxymatrine treatment, whereas Bcl-2
expression was upregulated in the oxymatrine-treated group.
In addition to rapid proliferation, tumor cells are character-
ized by metastasis by the degradation of the extracellular
matrix (ECM).25 MMP-2 can degrade the basement membrane
of the ECM to allow tumor cells to migrate out and accelerate
malignant progression.26 Studies have shown that oxymatrine
can reduce MMP9 expression in gastric cancer cells.27
Similarly, we showed that oxymatrine restrained the migration
and invasionofMCF-7 cellssignificantly alongwith thedown-
regulation of MMP9 expression. MMP9 is also downstream of
PI3K/Akt, so we conjectured that oxymatrine repressed the
metastasis of BC cells via the PI3K/Akt/MMP9 signaling
pathway. Xie W and colleagues reported that oxymatrine can
enhance the anti-tumor effects of bevacizumab against triple-
negative BC by abating the Wnt/β-catenin signaling
pathway.28 Our study demonstrated that oxymatrine can also
inhibit the growth of BC cells by regulating PI3K expression.
Figure 5 Suppression of proliferation and invasion and enhanced apoptosis of breast cancer cells by inhibition of expression of PI3K and its downstream signaling
pathway by oxymatrine.
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Conclusion Oxymatrine can limit the proliferation, apoptosis, migration
and invasion of BC cells by inhibiting expression of PI3K
and its downstream signaling pathways (Figure 5). However,
animal models of BC are needed to certify the validity and
safety of oxymatrine, and pharmacokinetic investigations
are needed to establish its optimal dose before clinical use
can be contemplated. We believe that oxymatrine could
serve as a potential therapeutic agent and deserves further
studies regarding anti-tumor treatment.
Author Contributions All authors contributed to data analysis, drafting or revising
the article, gave final approval of the version to be published,
and agree to be accountable for all aspects of the work.
Disclosure The authors report no conflicts of interest in this work.
References 1. Leone BA, Vallejo CT, Romero AO, et al. Prognostic impact of meta-
static pattern in stage IV breast cancer at initial diagnosis. Breast Cancer Res Treat. 2017;161(3):537–548. doi:10.1007/s10549-016-4066-7
2. IARC, WHO. Global cancer observatory; 2018. Available from: http://gco.iarc.fr/today/home. Accessed November 09, 2019.
3. Yoo TG, Cranshaw I, Broom R, et al. Systematic review of early and long-term outcome of liver resection for metastatic breast cancer: is there a survival benefit? Breast. 2017;32:162–172. doi:10.1016/j. breast.2017.02.003
4. Muhammad N, Steele R, Isbell TS, et al. Bitter melon extract inhibits breast cancer growth in preclinical model by inducing autophagic cell death. Oncotarget. 2017;8:66226–66236. doi:10.18632/oncotarget.19887
5. Bhattacharya S, Muhammad N, Steele R, et al. Bitter melon enhances natural killer-mediated toxicity against head and neck cancer cells. Cancer Prev Res (Phila). 2017;10(6):337–344. doi:10.1158/1940- 6207.CAPR-17-0046
6. Bhattacharya S, Muhammad N, Steele R, et al. Immunomodulatory role of bitter melon extract in inhibition of head and neck squamous cell carcinoma growth. Oncotarget. 2017;7(22):33202–33209. doi:10.18632/oncotarget.8898
7. Wang X, Liu C, Wang J, et al. Oxymatrine inhibits the migration of human colorectal carcinoma RKO cells via inhibition of PAI-1 and the TGF-β1/Smad signaling pathway. Oncol Rep. 2017;37 (2):747–753. doi:10.3892/or.2016.5292
8. Chen H, Zhang J, Luo J, et al. Antiangiogenic effects of oxymatrine on pancreatic cancer by inhibition of the NF-κB-mediated VEGF signaling pathway. Oncol Rep. 2013;30(2):589–595. doi:10.3892/ or.2013.2529
9. Zou J, Ran ZH, Xu Q, et al. Experimental study of the killing effects of oxymatrine on human colon cancer cell line SW1116. Chin J Dig Dis. 2005;6(1):15–20. doi:10.1111/j.1443-9573.2005.00181.x
10. Liu Y, Bi T, Dai W, et al. RETRACTED: effects of oxymatrine on the proliferation and apoptosis of human hepatoma carcinoma cells. Technol Cancer Res Treat. 2016;15(3):487–489. doi:10.1177/1533034615587616
11. Guo B, Zhang T, Su J, et al. Oxymatrine targets EGFR(p-Tyr845) and inhibits EGFR-related signaling pathways to suppress the prolifera- tion and invasion of gastric cancer cells. Cancer Chemother Pharmacol. 2015;75(2):353–363. doi:10.1007/s00280-014-2651-1
12. Zhang Y, Sun S, Chen J, et al. Oxymatrine induces mitochondria dependent apoptosis in human osteosarcoma MNNG/HOS cells through inhibition of PI3K/Akt pathway. Tumour Biol. 2014;35 (2):1619–1625. doi:10.1007/s13277-013-1223-z
13. Safdari Y, Khalili M, Ebrahimzadeh MA, et al. Natural inhibitors of PI3K/AKT signaling in breast cancer: emphasis on newly-discovered molecular mechanisms of action. Pharmacol Res. 2015;93:1–10. doi:10.1016/j.phrs.2014.12.004
14. Chia S, Gandhi S, Joy AA, et al. Novel agents and associated toxicities of inhibitors of the pi3k/Akt/mTOR pathway for the treatment of breast cancer. Curr Oncol. 2015;22(1):33–48. doi:10.3747/co.22.2393
15. Mayer IA, Arteaga CL. The PI3K/AKT pathway as a target for cancer treatment. Annu Rev Med. 2016;67:11–28. doi:10.1146/ annurev-med-062913-051343
16. Tian F, Ding D, Li D. Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells. Int J Oncol. 2015;46(6):2355–2363. doi:10.3892/ijo.2015.2959
17. Casimiro MC, Velasco-Velázquez M, Aguirre-Alvarado C, et al. Overview of cyclins D1 function in cancer and the CDK inhibitor landscape: past and present. Expert Opin Investig Drugs. 2014;23 (3):295–304. doi:10.1517/13543784.2014.867017
18. Xing Z, Zhang Y, Zhang X, et al. Fangchinoline induces G1 arrest in breast cancer cells through cell-cycle regulation. Phytother Res. 2013;27(12):1790–1794. doi:10.1002/ptr.4936
19. Takahashi-Yanaga F, Sasaguri T. GSK-3β regulates cyclin D1 expres- sion: a new target for chemotherapy. Cell Signal. 2008;20(4):581–589. doi:10.1016/j.cellsig.2007.10.018
20. Shalini S, Dorstyn L, Dawar S, et al. Old, new and emerging functions of caspases. Cell Death Differ. 2015;22(4):526–539. doi:10.1038/ cdd.2014.216
21. Zeng C, Ke Z, Song Y, et al. Annexin A3 is associated with a poor prognosis in breast cancer and participates in the modula- tion of apoptosis in vitro by affecting the Bcl-2/Bax balance. Exp Mol Pathol. 2013;95(1):23–31. doi:10.1016/j.yexmp.2013.04.002
22. Sharifi S, Barar J, Hejazi MS, et al. Doxorubicin changes Bax/Bcl-xL ratio, caspase-8 and 9 in breast cancer cells. Adv Pharm Bull. 2015;5 (3):351–359. doi:10.15171/apb.2015.049
23. Vafaiyan Z, Gharaei R, Asadi J. The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acid-treated MCF-7 breast cancer cell line. Iran J Basic Med Sci. 2015;18(7):700–704.
24. Kordezangeneh M, Irani S, Mirfakhraie R, et al. Regulation of BAX/ BCL2 gene expression in breast cancer cells by docetaxel-loaded human serum albumin nanoparticles. Medical Oncology. 2015;32 (7). doi:10.1007/s12032-015-0652-5
25. Das S, Banerji A, Frei E, et al. Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium. Life Sci. 2008;82(9–- 10):467–476. doi:10.1016/j.lfs.2007.12.013
26. Nilsson UW, Garvin S, Dabrosin C. MMP-2 and MMP-9 activity is regulated by estradiol and tamoxifen in cultured human breast cancer cells. Breast Cancer Res Treat. 2007;102(3):253–261. doi:10.1007/ s10549-006-9335-4
27. Guo B, Zhang T, Su J, et al. Oxymatrine targets EGFRp-Tyr845 and inhibits EGFR-related signaling pathways to suppress the proliferation and invasion of gastric cancer cells. Cancer Chemother Pharmacol. 2017;75(2):353–363. doi:10.1007/s00280-014-2651-1
28. Xie W, Zhang Y, Zhang S, et al. Oxymatrine enhanced anti-tumor effects of Bevacizumab against triple-negative breast cancer via abating Wnt/β- Catenin signaling pathway. Am J Cancer Res. 2019;9(8):1796–1814.
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