Sinensetin (SIN) is a polymethoxy flavone primarily present in citrus fruits. This compound has demonstrated anticancer activity. However, the underlying mechanism of its action has not been fully understood. The present study investigated the impact of SIN on angiogenesis in a liver cancer model. In a murine xenograft tumor model, SIN inhibited the growth of HepG2/C3A human liver hepatoma cell-derived tumors and reduced the expression levels of platelet/endothelial cell adhesion molecule-1 and VEGF. In HepG2/C3A cells, SIN repressed VEGF expression by downregulating hypoxia-inducible factor expression. In cultured human umbilical vein endothelial cells, SIN increased apoptosis and repressed migration and tube formation. In addition, SIN decreased the phosphorylation of VEGFR2 and inhibited the AKT signaling pathway. Molecular docking demonstrated that the VEGFR2 core domain effectively combined with SIN at various important residues. Collectively, these data suggested that SIN inhibited liver cancer angiogenesis by regulating VEGF/VEGFR2/AKT signaling.
Angiogenesis is required for tumor development (
Sinensetin (SIN) is a polymethoxy flavone containing five methoxy groups that is present mainly in citrus fruits (
Angiogenesis is regulated by various angiogenic factors, such as VEGF and its receptor (
The present study observed that SIN inhibited the growth and angiogenesis of HepG2/C3A-derived tumors
The antibody against VEGF was purchased from Novus Biologicals. The primary antibodies including anti-VEGFR2 (cat. no. 9698), anti-phosphorylated (p)-VEGFR2 (Tyr1175) (cat. no. 3770), anti-AKT (cat. no. 4691), anti-p-AKT (Ser473) (cat. no. 4060), anti-platelet/endothelial cell adhesion molecule-1 (CD31) (cat. no. 77699), β-actin (cat. no. 4970), GAPDH (cat. no. 2118) and α-tubulin (cat. no. 2125) were acquired from Cell Signaling Technology, Inc.; anti-HIF-1α (cat. no. 20960-1-AP) was provided by ProteinTech Group, Inc. Synthetic SIN (>98% purity; cat. no. SS8550) was purchased from Beijing Solarbio Science & Technology Co. Ltd. and characterized by mass spectrometry. Recombinant human VEGF (VEGF165; cat. no. 100-20) and insulin-like growth factor 1 (IGF-1; cat. no. 100-11) were purchased from PeproTech, Inc. SU1498 (cat. no. HY-19326) is a selective VEGFR2 inhibitor, which was obtained from MedChemExpress.
A total of 10 male BALB/c nude mice (age, 4 weeks; weight, 15-20 g) purchased from Beijing Weitong Lihua Biotechnology Co., Ltd., were used for the tumor xenograft growth assay. Mice were housed at room temperature (22±1˚C) with 50% humidity under special pathogen-free conditions with a 12-h light/dark schedule. All animal studies were conducted with approval (no. 2017-106) obtained from the Ethics Committee of Shandong Provincial Qianfoshan Hospital. HepG2/C3A cells (ATCC) (5x107/ml) suspended in 0.2 ml 1:1 serum-free DMEM and Matrigel (Corning, Inc.) were implanted into the right flank of the mice (
The tumors were fixed overnight in 4% formaldehyde at room temperature, and then ethanol was used for dehydration at the conventional gradient, xylene for vitrification and paraffin for embedding. Sections (5 µm) were created and deparaffinized with graded xylene and rehydrated by graded ethanol. Following heat-induced antigen retrieval in citrate buffer (pH 6.0) in a high-pressure sterilizer at 121˚C for 10 min, the slides were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity and subsequently incubated with 4% bovine serum albumin at 37˚C for 30 min. The tumor tissue slides were incubated with primary anti-CD31 antibodies (1:100) at 4˚C overnight and washed with PBS three times. HRP-labeled secondary antibody from the MaxVision™ HRP-Polymer anti-mouse/rabbit IHC kit (ready to use; cat. no. KIT5020; Fuzhou Maixin Biotech Co., Ltd.) was applied and incubated for 30 min at room temperature. The slides were then dehydrated in an ascending graded series of absolute ethyl alcohols, cleared in xylene and cover-slipped with neutral balsam. Following treatment with hematoxylin for 2 min at room temperature to stain the nuclei, images were captured using a light microscope and in ten random fields at x200 magnification.
HUVECs (cat. no. PCS-100-010) and HepG2/C3A (cat. no. CRL-10741) cells were purchased from the American Type Culture Collection. HUVECs were incubated in Endothelial Cell Medium (ECM; ScienCell Research Laboratories, Inc.) containing 5% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% EC growth supplement (ECGS; ScienCell Research Laboratories, Inc.) at 37˚C. HepG2/C3A cells were incubated in Dulbecco's modified Eagle's medium (HyClone; Cytiva) comprising 10% FBS in a hypoxic incubator with 94% N2, 5% CO2 and 1% O2 at 37˚C.
HUVECs were harvested and seeded into 96-well plates at a density of 5x103 cells/well and exposed to various concentrations of SIN (3, 10, 30, 60 and 100 µM). Following culture for 24 h, the viability of HUVECs was detected using CCK-8 (Dojindo Molecular Technologies, Inc.). The optical density, which represented the proliferation of HUVECs, was measured at 450 nm using a Spectra Max 190 (Molecular Devices, LLC).
The induction of apoptosis in HUVECs was assessed using Annexin V-FITC and propidium iodide staining (ELabscience Biotechnology, Inc.). Following treatment with SIN, HUVECs were collected and resuspended in a binding buffer at a final concentration of 1x106 cells/ml. Single cells were incubated with 5 µl Annexin V-FITC and 5 µl PI for 15 min at room temperature in the dark. The percentages of early and late apoptotic cells were assessed using a FACSAria II flow cytometer (BD Biosciences) to calculate the apoptotic rate. Data were analyzed using FlowJo (V10; FlowJo LLC).
Migration was assessed using a scratch wound healing assay. The cells were seeded (4x105/well) into 6-wellplates. A straight scratch was introduced in HUVEC monolayers using a 200-µl plastic pipette tip. Following incubation for a further 24 h in EBM containing 1% FBS, the average distance of the cells migrating into the wound was monitored by a light microscope (magnification, x100; Olympus Corporation). The migrated distance was calculated using ImageJ software (version1.49p; National Institutes of Health).
Transwell inserts (Corning, Inc.) with a pore size of 8-µm were used to assess the migratory ability of ECs. HUVECs (1x105/well) were resuspended in 500 µl serum-free medium with SIN (30 µM) and subsequently added to the upper plate compartment. The medium supplemented with 10% FBS was filled to the bottom chamber. The invaded cells were treated with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet (Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Finally, the cells numbers in five randomly selected fields were counted under an inverted microscope (magnification, x100; Olympus Corporation).
For this assay, 96-well plates were pre-coated with Matrigel (50 µl) for 30 min at 37˚C. SIN was dissolved in DMSO, which was used as a control group. HUVECs were resuspended at 1-2x104 cells/ml in serum-free EBM-2 with SIN and loaded on top of the Matrigel. Following culture for 6 h, the images were captured using a light microscope (magnification, x40; Olympus Corporation). Vessel morphometric parameters, including vessel number, were quantified using ImageJ software (version1.49p; National Institutes of Health). Tube formation was expressed as a percentage of the control group.
The lysates of HUVECs were extracted using RIPA lysis buffer. The homogenates were centrifuged at 12,000 x g for 15 min at 4˚C. The concentration levels of the protein samples were evaluated using a bicinchoninic acid protein analysis kit (Thermo Fisher Scientific, Inc.). Total protein (30 µg) was electrophoresed on 7.5 and 10% SDS-PAGE gels for 1 h using an electrophoresis apparatus (Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes (MilliporeSigma) at 280 mA for 2 h, followed by blocking in 5% non-fat milk for 1.5 h. The membranes were incubated at 4˚C overnight with rabbit CD31, p-VEGFR2, VEGFR2, p-AKT, AKT, β-actin and GAPDH (1:1,000 each), HIF-1α (1:500), α-tubulin (1:3,000) and mouse VEGF (1:1,000) antibodies. Following washing with TBS-T (0.1% Tween-20) three times, the membranes were incubated with HRP-conjugated secondary antibodies (ShanghaiMorui Biotechnology) for 2 h at room temperature and developed with enhanced chemiluminescence (ECL; MilliporeSigma) reagents. The signal intensity was calculated using ImageJ software (version1.44p; National Institutes of Health).
HepG2/C3A cells were plated at a density of 5x105 cells/ml in 6-well plates with DMEM containing 10% FBS overnight. At 90% confluence, the cells were transferred from a normoxic (21% O2) to a hypoxic (1% O2) environment for 48 h following replacement of the medium with or without 30 µM SIN. The collected CM was filtered through a 0.2-µm filter (Corning, Inc.) and stored in a freezer at -80˚C.
VEGF levels in HepG2/C3A CM were assessed using a human VEGF Quantikine ELISA kit (cat. no. E-TSEL-H0026; ELabscience Biotechnology, Inc.) following the manufacturer's protocol. The experiment was repeated in triplicate, with five biological samples each time.
HepG2/C3A cells (2x105/ml) were treated with or without SIN in 6-well plates under hypoxic conditions for 48 h. Total RNA in the cells was extracted using RNAiso Plus kit (Takara Bio, Inc.). The RNA concentration levels were measured using spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific, Inc.). Following treatment with DNAse, 1 µg RNA was reverse-transcribed using a reverse transcriptase kit (Thermo Fisher Scientific, Inc.). RT-qPCR was performed using SYBR-Green I Master (Vazyme Biotech Co., Ltd.). RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. The primer sequences used were obtained from a previously published study (
The crystal structure file of VEGFR2 (PDB ID: 3VHE) was downloaded from the Protein Data Bank (PDB) database (
All results are presented as the mean ± standard deviation (SD). Unpaired Student's t-test was applied for two-group comparisons. Significance among multiple groups were calculated by one-way ANOVA with Bonferroni's post-hoc test using GraphPad Prism 5 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of SIN is displayed in
Under different SIN concentrations (3, 10, 30, 60 and 100 µM) for 24 h, cytotoxicity was measured using CCK8 assay. VEGF was used as a positive control in the present study. As an angiogenic factor secreted by tumor cells under hypoxic conditions (
VEGF is a pivotal enabling factor for angiogenesis (
VEGF binds to VEGF receptor 2 on ECs and promotes proliferation, migration and survival of ECs (
To further demonstrate the anti-angiogenesis mechanism of SIN, whether it depended on p-VEGFR2, p-AKT, or both, VEGF and SU1498 were used in a serum-free medium to treat HUVECs. As a selective inhibitor of VEGFR2, SU1498 was used as a positive control (
As one of the best theoretical methods, molecular docking has traditionally been employed to study binding affinities between target proteins and virtually screened ligands (
Chinese herbs are an essential part of Traditional Chinese Medicine and contribute to liver cancer management in China (
In the present study, an
Angiogenesis requires EC proliferation and migration to appropriate positions leading to their assembly in vascular structures (
Angiogenesis is necessary for tumor development and provides oxygen and nutrients to tumor cells (
VEGF binds to VEGFR2 on the EC membrane and initiates angiogenesis (
VEGFR2 phosphorylation activates downstream signaling pathways, such as MAPK/ERK and PI3K/AKT (
The current study contains certain limitations. Although it was shown that the expression levels of the HIF-1α protein were downregulated by SIN, the detailed mechanism of this process remains unclear. The translation of the HIF-1α protein is considered to be an important regulatory mechanism of HIF-1α-inhibiting compounds (
In summary, the present study demonstrated that SIN exhibited a significant antiangiogenic effect by inhibiting the viability of ECs and inducing apoptosis. Concomitantly, SIN suppressed angiogenesis by inhibiting the migratory activity and tube formation in HUVECs. SIN potently inhibited angiogenesis
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JLiu designed the experiments. XL, YLi and YWa performed the experiments and prepared the article. FL, YLiu, YWu, HR and XZ performed the animal experiments and analysis. JLia and RZ participated in data analysis discussions. XL and JLiu confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
All animal experiments and experimental protocols were approved by the Ethics Committee of Qianfoshan Hospital (Jinan, China; approval no. 2017-106; approval date: 14 December 2017).
Not applicable.
The authors declare that they have no competing interests.
Effects of SIN on HepG2/C3A tumor growth and angiogenesis in BALB/c nude mice. (A) Chemical structure of SIN. (B) Representative images of HepG2/C3A cell-derived tumors obtained on the last day of the animal experiment. (C) Growth curve of HepG2/C3A cell-derived tumors. n=5, *P<0.05 vs. the control group. (D) Body weight change of the different groups. n=5. (E) Representative images of IHC staining (magnification, x200) in tumor samples on day 14 and statistical analysis of CD31 staining results. n=3, *P<0.05 vs. the control group. (F and G) Western blotting and relative gray value analyses of CD31 and VEGF protein expressions in tumor tissues. n=3, **P<0.01. SIN, sinensetin; IHC, immunohistochemical; CD31, platelet/endothelial cell adhesion molecule-1; VEGF, vascular endothelial growth factor; AOD, average optical density.
SIN inhibits angiogenesis
SIN suppresses angiogenesis by inhibiting the activity of VEGF in HepG2/C3A cells. (A) SIN down regulated the expression levels of VEGF and HIF-1α under hypoxic conditions as determined by western blot analysis. n=3, *P<0.05. (B) SIN inhibits VEGF secretion of HepG2/C3A cells under hypoxic conditions as determined by ELISA. n=5, *P<0.05, ***P<0.001. (C) The expression levels of VEGF were analyzed by RT-qPCR in HepG2/C3A cells. n=3, *P<0.05, **P<0.01. (D) HepG2/C3A cells were pre-incubated with SIN for 48 h in a hypoxic environment and subsequently CM was collected to assess tube formation. SIN-treated CM inhibits the tube-formation ability of HUVEC (magnification, x40). n=8, ***P<0.001. SIN, sinensetin; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor 1α; RT-qPCR, reverse transcription-quantitative PCR; CM, tumor-conditioned medium.
SIN inhibits phosphorylation of VEGFR2 and AKT in HUVECs. (A) SIN inhibits the phosphorylation of VEGFR2 induced by VEGF. The expression levels of p-VEGFR2 and VEGFR2 in HUVECs treated with SIN were analyzed by western blotting. n=3, *P<0.05, **P<0.01. (B) SIN inhibited VEGFR2-induced phosphorylation of AKT. p-AKT and AKT were examined in HUVECs following treatment with SIN. n=3, *P<0.05, **P<0.01. SIN, sinensetin; VEGFR, vascular endothelial growth factor receptor; HUVECs, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; p-, phosphorylated.
Decrease in AKT phosphorylation levels induced by SIN is dependent on the decrease of VEGFR2 phosphorylation. (A) The inhibitory effect of SIN on VEGFR2 phosphorylation was similar to that of SU1498. n=3, ***P<0.001. (B) SIN inhibits VEGFR2-induced phosphorylation of AKT, which is reversible by IGF-1. n=3, *P<0.05, **P<0.01. SIN, sinensetin; VEGFR, vascular endothelial growth factor receptor; IGF-1, insulin-like growth factor 1.
Optimal conformations between SIN and VEGFR2 are estimated by molecular docking. (A) The binding modes of the SIN-VEGFR2 complex with minimum energy. (B) The overall interactions between SIN and the protein residues. (C) The optimal binding position of SIN with VEGFR2. (D) Representation of the interaction between SIN and VEGFR2. SIN, sinensetin; VEGFR, vascular endothelial growth factor receptor.
Schematic diagram summarizing the signaling pathway by which SIN inhibits liver cancer angiogenesis. SIN represses VEGF expression by downregulating HIF-1α expression. SIN inhibits VEGF-induced VEGFR2 phosphorylation and sequentially inhibits the levels of p-AKT leading to inhibition of EC proliferation, migration, tube formation and angiogenesis. SIN, sinensetin; VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factors; VEGFR, vascular endothelial growth factor receptor; EC, endothelial cell; p, phosphorylated.