Contributed equally
Ophiopogonin-B (OP-B) is a bioactive component from the root of
Liver cancer is a leading cancer that occurs most commonly and is considered as the fourth most prevalent human tumor globally (
Traditional Chinese medicine (TCM) is an important therapy for liver cancer in China. Owing to its unique overall concept, treatment based on syndrome differentiation and abundant natural medicine resources, it has become a characteristic method throughout the entire process of liver cancer prevention and treatment (
Protein tyrosine phosphatase 1B [PTP1B; encoded by protein tyrosine phosphatase non-receptor type 1 (PTPN1)] is an important member in PTP superfamily of proteins. PTPs catalyze phosphate monoesters hydrolysis on tyrosine residues; they are also known to act as signaling molecules to regulate a number of cellular processes, including cell growth, differentiation, mitotic cycle and oncogenic transformation (
The human normal hepatocyte cell line HHL-5 and the HCC cell line MHCC97-H were obtained from the American Type Culture Collection. Both cell lines were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS; Wisent Biotechnology) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) in an incubator with 5% CO2 at 37°C. For OP-B treatment, cells were cultured in DMEM containing different concentrations (5, 10, 20 and 40 µM) of OP-B (purity >97%; Shanghai Yuanye Bio-Technology Co., Ltd.) for 24 h as previously described (
A PTP1B overexpression vector (Ov-PTP1B) was constructed by cloning human PTP1B cDNA into the pcDNA3.1 vector (Thermo Fisher Scientific, Inc.). Empty pcDNA3.1 vector was used as negative control (Ov-NC). MHCC97-H cells were grown in six-well plates and subsequently transfected with 2 µg of either vector using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h according to the manufacturer's protocol. At 48 h post-transfection, monoclonal cells were then selected and examined for PTP1B overexpression.
A total of 15 male BALB/c nude mice (age, 4–5 weeks; 15–20 g; Shanghai Laboratory Animal Center) were maintained under specific pathogen-free conditions at room temperature under a controlled 12/12 h light/dark cycle, and received food and water
Xenografted tumor tissues were fixed with 4% paraformaldehyde at 4°C for 12 h and embedded in paraffin. After being deparaffinized with xylene and rehydrated with a descending ethanol series, slides (3 µm thickness) were stained with the IHC Assay Kit (cat. no. KGOS300; Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol and then incubated at 4°C overnight with the following primary antibodies purchased from Abcam: Anti-Ki67 (1:1,000; cat. no. ab15580), anti-CD31 (1:50; cat. no. ab28364) and anti-VEGFA (1:200; cat. no. ab52917). Secondary antibody goat anti-rabbit IgG (1:1,000; cat. no. ab6721; Abcam) was applied at room temperature for 2 h. Images of the tissue sections were then captured with a fluorescence microscope (DMi8; Leica Microsystems GmbH; ×200 magnification).
Cell Counting Kit-8 (CCK8; Beyotime Institute of Biotechnology) was used to measure cell viability. Briefly, cells were seeded into 96-well plates at a density of 1,000 cells/well at 37°C for 24 h. After treatment with different concentrations of OP-B (0, 5, 10, 20 and 40 µM) at 37°C for 24 h, cells were incubated with 10 µl of CCK-8 solution at 37°C for 2 h. The optical density was calculated at 450 nm using a microplate reader (Model550; Bio-Rad Laboratories, Inc.).
For the colony formation assay, 1×103 MHCC97-H cells were cultivated in six-well plates in a 37°C incubator with 5% CO2. After 14 days, cell colonies were fixed with 95% alcohol at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min (Sigma-Aldrich; Merck KGaA). Images were captured with a light microscope (Nikon Corporation; ×200 magnification) and colonies were counted using ImageJ software (version 1.8.0; National Institutes of Health).
According to the manufacturer's protocol, MHCC97-H cell apoptosis was analyzed using TUNEL Assay kit (Abcam). Briefly, cells were fixed in 4% paraformaldehyde solution for 30 min at room temperature, treated with 0.2% Triton X-100 for 5 min at room temperature, washed twice in PBS at room temperature, and labeled with fluorescein-12-dUTP using terminal deoxynucleotidyl transferase for 2 h at room temperature. Subsequently, cell nuclei were stained with 5 µg/ml DAPI at room temperature for 3 min. All images were obtained using a fluorescence microscope (DMi8; Leica Microsystems GmbH; ×200 magnification).
Briefly, MHCC97-H cells were seeded in a six-well plate at a density of 5×105 cells/well in DMEM. When the cells were ~80% confluent, scratches were made in the middle of slides using a sterile 10-µl pipette tip. After incubation for another 48 h with or without 20 µM OP-B in serum-free DMEM at 37°C, images were captured to estimate closure of the gap using a light microscope (Nikon Corporation; ×200 magnification). Migration distance was evaluated using ImageJ software (version 1.8.0; National Institutes of Health) and calculated as follows: Migration distance=(width of gap at 0 h-width of gap at 48 h)/width of gap at 0 h. Data are shown as relative migration distance by normalization to the control group.
The invasive ability of MHCC97-H cells was evaluated by the Biocoat invasion assay kit (Corning, Inc.) strictly following the manufacturer's protocol. Firstly, 5×105 cells per well were plated in the upper chamber followed by treatment with 20 µM OP-B or not. After 12 h of treatment, the cells in the upper chamber were incubated with serum-free DMEM medium, whereas media supplemented with 10% FBS was placed at the lower chamber. After 48 h, the invasive cells at the lower chamber were stained with 0.1% crystal violet at room temperature and observed using an optical light microscope (Leica Microsystems GmbH; ×200 magnification).
Matrigel (BD Biosciences) was thawed at 4°C, pipetted into 96-well plates and allowed to polymerize at 37°C for 1 h. Cells (1×104 per well) were suspended in DMEM containing 0 or 20 µM OP-B and seeded onto the Matrigel. After incubation for 24 h at 37°C, tube formation was analyzed by counting nodes and measuring total tube numbers using ImageJ software (version 1.8.0; National Institutes of Health).
Total RNA was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and then reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. SYBR-Green Supermix (Takara Biotechnology Co., Ltd.) was used for qPCR in a 10 µl reaction volume on the Roche Light Cycler R480 System (Roche Diagnostics). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 70°C for 10 sec. Target gene expression levels were analyzed using the 2−ΔΔCq (
Total protein was extracted from cells or tumor tissues by RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA Kit (Beyotime Institute of Biotechnology), mixed with 5X sample buffer and boiled at 95°C for 5 min. Equal amounts (40 µg) of protein lysate per sample were separated on 10% gels using SDS-PAGE and transferred onto the PVDF membranes. After blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies (goat anti-rabbit IgG HRP; 1:10,000; cat. no. ab6721; Abcam) at room temperature for 2 h. An enhanced chemiluminescence (ECL) detection kit (Amersham; Cytiva) was used to visualize the protein bands. Band intensity was semi-quantified by ImageJ software (v1.8.0; National Institutes of Health). Primary antibodies (all from Abcam) included: anti-PTP1B (1:1,000; cat. no. ab252928), anti-Bcl-2 (1:1,000; cat. no. ab32124), anti-Bax (1:5,000; cat. no. ab32503), anti-cleaved caspase 3 (1:500; cat. no. ab32042), anti-caspase-3 (1:1,000; cat. no. ab184787), anti-cleaved poly ADP-ribose polymerase (PARP; 1:5,000; cat. no. ab32064), anti-PARP (1:5,000; cat. no. ab191217), anti-E-cadherin (1:10,000; cat. no. ab40772), anti-N-cadherin (1:10,000; cat. no. ab76011), anti-Vimentin (1:1,000; cat. no. ab45939), anti-VEGFA (1:10,000; cat. no. ab52917), anti-phosphorylated (p)-phosphatidylinositol 3 kinase (PI3K; 1:1,000; cat. no. ab191606), anti-total (t)-PI3K (1:1,000; cat. no. ab278545), anti-p-AKT (1:1,000; cat. no. ab38449), anti-t-AKT (1:1,000; cat. no. ab8805), anti-p-adenosine 5′-monophosphate-activated protein kinase (AMPK; 1:1,000; cat. no. ab109402), anti-t-AMPK (1:1,000; cat. no. ab214425) and anti-GAPDH (1:10,000; cat. no. ab181602).
Data are expressed as the mean ± standard deviation. One-way ANOVA followed by Tukey's post hoc test was used for analysis between multiple groups. Calculations were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
The anticancer effects of OP-B on HCC were first investigated
To validate the effect of OP-B on HCC and PTP1B expression
Subsequently, PTP1B was overexpressed in MHCC97-H cells by transfection of Ov-PTP1B vector and then the overexpression efficiency was confirmed by RT-qPCR and western blotting (
To observe the effect of OP-B on the malignant processes of HCC, MHCC97-H cells overexpressing PTP1B were treated with 20 µM OP-B, then cell proliferation, apoptosis, migration, invasion and angiogenesis were evaluated. As revealed in
Results from wound healing, invasion and tube formation assays demonstrated that OP-B significantly inhibited MHCC97-H cell migration, invasion and angiogenesis compared with untreated control cells (
To uncover the underlying related signaling mechanism of OP-B, the expression of the PI3K/AKT and AMPK signaling pathways was detected. As demonstrated in
OP-B is a natural active compound extracted from the TCM
Natural compounds from TCM have been revealed to exert remarkable effects in the treatment of HCC (
To uncover the mechanism through which OP-B exert its anticancer effect, its potential downstream targets were predicted using SwissTargetPrediction online database and PTP1B was identified as one of the targets of OP-B (data not shown). Notably, the participation of PTP1B in liver cancer initiation and progression has been previously reported (
Finally, the mechanisms underlying the actions of OP-B/PTP1B in HCC were uncovered. Jin
Collectively, the present study demonstrated that OP-B suppressed the progression of HCC both
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JS and YZ conceived and designed the study. FY, QG and HT performed the experiments. FY and QG analyzed and interpreted the data. JS and YZ drafted the manuscript and revised it for critically important intellectual content. JS and YZ confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The animal study protocol was approved by the Ethics Committee of Suzhou Hospital of Integrated Traditional Chinese and Western Medicine (Suzhou, China; approval no. 20180901).
Not applicable.
The authors declare that they have no competing interests.
Effect of OP-B on HCC progression and PTP1B expression
Effect of OP-B on PTP1B expression in hepatocellular carcinoma cells. (A) HHL-5 and MHCC97-H cells were exposed to different concentrations of OP-B for 24 h, then cell viability was measured by Cell Counting Kit-8 assay. MHCC97-H cells were treated with different concentrations of OP-B for 24 h, then PTP1B (B) mRNA and (C) protein expression levels were evaluated. MHCC97-H cells were either transfected with Ov-PTP1B or empty vector, then the overexpression efficiency was verified by (D) reverse transcription-quantitative PCR and (E) western blot assays. *P<0.05, **P<0.01 and ***P<0.001 vs. control. NC, negative control; OP-B, ophiopogonin-B; Ov, overexpression vector; PTP1B, protein tyrosine phosphatase 1B.
Effect of OP-B and PTP1B overexpression on hepatocellular carcinoma cell proliferation and apoptosis. MHCC97-H cells were transfected with Ov-PTP1B or OV-NC and subsequently treated with 20 µM OP-B. (A) Cell viability was measured by Cell Counting Kit-8 assay. (B) Colony formation was performed to observe cell proliferation. (C) TUNEL staining was used to assess apoptosis. (D) Protein expression levels of apoptosis-related proteins were detected by western blotting; GAPDH was used as the loading control. ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. OP-B + Ov-NC. NC, negative control; OP-B, ophiopogonin-B; Ov, overexpression vector; PTP1B, protein tyrosine phosphatase 1B.
Effect of OP-B treatment and PTP1B overexpression on migration, invasion and angiogenesis of hepatocellular carcinoma cells. MHCC97-H cells were transfected with Ov-PTP1B or OV-NC and subsequently treated with 20 µM OP-B. (A) Cell migration was determined by wound healing assay. (B) Transwell assay was performed to observe cell invasion. (C) Tube formation assay was used to assess cell angiogenesis. (D) Protein expression levels of E-cadherin, N-cadherin, Vimentin and VEGFA were detected by western blot analysis. ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. OP-B + Ov-NC. NC, negative control; OP-B, ophiopogonin-B; Ov, overexpression vector; PTP1B, protein tyrosine phosphatase 1B.
Effect of OP-B and PTP1B overexpression on PI3K/AKT and AMPK signaling in hepatocellular carcinoma cells. MHCC97-H cells were transfected with Ov-PTP1B or OV-NC and subsequently treated with 20 µM OP-B, then the protein expression levels of components of the PI3K/AKT and AMPK signaling pathways were detected by western blotting. ***P<0.001 vs. control; ###P<0.001 vs. OP-B + Ov-NC. NC, negative control; OP-B, ophiopogonin-B; Ov, overexpression vector; PTP1B, protein tyrosine phosphatase 1B.