Lung cancer is one of the most common types of cancer in the world, resulting in numerous cancer-associated deaths. The properties of cancer stem cells (CSCs) are important for the initiation and deterioration of lung cancer. Schisandrin B (SchB), an active compound extracted from
Lung cancer is regarded as one of the leading causes of cancer-associated mortality worldwide, accounting for ~18.4% of cancer-associated deaths (
Cancer stem cells (CSCs) are well-known heterogeneous tumor cells characterized by the capacity to self-renew, indefinitely proliferate, differentiate and initiate tumors; these properties are considered as the promoting factors for tumorigenesis, metastasis and recurrence (
It is widely acknowledged that epithelial-mesenchymal transition (EMT) is defined as a cellular programme with representative changes of cell phenotype from an epithelial to a mesenchymal morphology, which is strongly associated with the acquisition of CSC properties (
Schisandrin B (SchB) is the most active dibenzocyclooctadiene derivative extracted from the traditional Chinese herb
SchB extracted from
Human large-cell lung cancer cell lines, NCI-H460 and H661, were supplied by the American Type Culture Collection. Cells were grown in RPMI-1640 medium containing 10% FBS (both HyClone; Cytiva) under an atmosphere of 5% CO2 in a sterile incubator at 37°C.
To establish cancer stem-like cells (named NCI-H460-CSC or H661-CSC), NCI-H460 or H661 cells were maintained for 10 days at 37°C in serum-free DMEM/F12 medium (Sigma-Aldrich; Merck KGaA) complemented with 100 µg/ml insulin (Sigma-Aldrich; Merck KGaA), 20 ng/ml epidermal growth factor (Invitrogen; Thermo Fisher Scientific, Inc.), 20 ng/ml basic fibroblast growth factor (Invitrogen; Thermo Fisher Scientific, Inc.), 5 mM Hepes (Sigma-Aldrich; Merck KGaA), 0.6% glucose (Sigma-Aldrich; Merck KGaA) and 0.4% BSA (Sigma-Aldrich; Merck KGaA). The media was changed every 2 days. CD133+ cells (cancer stem-like cells) in induced cells were isolated by magnetic-activated cell sorting for further experiments. According to the manufacturer's instructions, cells were labeled using CD133-PE antibody (Miltenyi Biotec GmbH; 1:50; cat. no. 130-113-748) at 4°C for 10 min in the dark and underwent magnetic sorting with the help of 10 µl anti-PE Multisort MicroBeads (Miltenyi Biotec GmbH; cat. no. 130-090-757) per 90 µl cell suspension. Flow cytometry was used to analyze CD133+ cells. In subsequent experiments, sorted cells (cancer stem-like cells) were incubated in serum-free DMEM/F12 medium containing the aforementioned growth factors.
TNF-α and anisomycin were supplied by R&D Systems and Invitrogen (Thermo Fisher Scientific, Inc.), respectively. To activate the NF-κB and MAPK signaling pathways, cells were incubated with 10 ng/ml TNF-α and 2 ng/ml anisomycin for 30 min at 37°C, respectively. For cells treated with SchB and TNF-α or anisomycin, cells were treated with 40 µmol/l SchB for 12 h at 37°C and then stimulated with 10 ng/ml TNF-α or 2 ng/ml anisomycin for 30 min at 37°C.
Cancer stem-like cells and parental cells were inoculated into 96-well plates at a density of 103 cells/well and cultured at 37°C. After reaching 80% confluence, cancer stem-like cells and parental cells were incubated with different doses of SchB (0, 5, 10, 20, 40, 80, 120 and 160 µmol/l) at 37°C. After 72 h of treatment with SchB, 10 µl CCK-8 reagent (MedChemExpress) was added for 2 h at 37°C. Subsequently, the optical density value at 450 nm was determined using a microplate reader (Molecular Devices, LLC).
Flow cytometry was used to examine the ratio of CD133+ cells according to the manufacturer's instructions (BD Biosciences). Following different treatments, the collected CSCs were rinsed with PBS, followed by incubation with a primary antibody against CD133 (Abcam; 1:100; cat. no. ab216323) at room temperature for 30 min and then probed with an Alexa Fluor594-labeled IgG secondary antibody (Abcam; 1:2,000; cat. no. ab150080) at 4°C in the dark for 30 min. The ratio of CD133+ cells was analyzed using a BD FACSCalibur™ flow cytometer (BD Biosciences) and FlowJo 7.6 software (FlowJo LLC).
Cell cycle was analyzed via flow cytometry after PI staining. In brief, cancer stem-like cells and parental cells were harvested and washed with PBS twice, followed by fixation using ice-cold 70% ethanol for 1 h at 4°C. Subsequently, cells were stored at −20°C. Before analysis, fixed cells were washed with PBS twice, and incubated in PBS containing 50 µg/ml PI (Beyotime Institute of Biotechnology), 0.3% Triton-X 100 and 100 µg/ml RNase A (Sigma-Aldrich; Merck KGaA) in the dark for 30 min at 37°C. Afterwards, ~3×104 events were subjected to flow cytometry analysis using a BD FACSCalibur™ flow cytometer (BD Biosciences), and the percentage of cells at different stages of the cell cycle was analyzed using the FlowJo 7.6 software.
Following treatment, 1×105 cancer stem-like cells were seeded in 6-well plates. When reaching 90% confluence, a sterile micropipette tip was used to scrape the cell monolayer to form the wound. Subsequently, the cell monolayer was washed with PBS and cultured with serum-free DMEM/F12 medium containing the aforementioned growth factors at 37°C. The distance of wound gap was monitored and photographed using an inverted light microscope (Nikon Corporation; magnification, ×100 and ×200) at 0 and 48 h following scratching.
The invasive capacity of CSCs was estimated using Transwell assays with Matrigel-coated Transwell inserts (Corning, Inc.). In brief, after treatment, 5×104 cells were seeded in the upper compartment containing 200 µl serum-free DMEM/F12 medium containing the aforementioned growth factors, while 600 µl complete DMEM/F12 medium (containing 10% FBS and the aforementioned growth factors) was added to the lower compartment. Following 24 h of incubation at 37°C, cells in the upper chamber were removed using a cotton swab. The invaded cells were immobilized in 4% paraformaldehyde at room temperature for 10 min, stained with 0.5% crystal violet at room temperature for 15 min and photographed in five randomly selected fields under an inverted light microscope (Nikon Corporation; magnification, ×100 and ×200).
After different treatments, 5×103 cancer stem-like cells were inoculated into each well of 6-well ultralow attachment plates (Corning, Inc.) and incubated at 37°C in serum-free DMEM/F12 medium containing the aforementioned growth factors for 10 days. The medium was replaced every other day. Images were obtained with an inverted light microscope (Nikon Corporation; magnification, ×400). Spheres that reached a diameter of 100 µm in five random fields were counted for one sample. Each group was replicated three times.
Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology). Tumor tissues were homogenized in RIPA buffer. Protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology). Equal amounts of proteins (50 µg/lane) were electrophoretically separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Subsequently, membranes were blocked with 5% skimmed milk, followed by incubation with primary antibodies overnight at 4°C and then incubation with appropriate HRP-labeled secondary antibodies (Abcam; 1:5,000; cat. nos. ab6721 and ab6728) at room temperature for 1 h. An enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was used to detect the protein signals. The specific primary antibodies included antibodies against E-cadherin (Abcam; 1:200; cat. no. ab219332), vimentin (Abcam; 1:1,000; cat. no. ab8978), N-cadherin (Abcam; 1:1,000; cat. no. ab245117), CD133 (Abcam; 1:1,000; cat. no. ab216323), CD44 (Abcam; 1:1,000; cat. no. ab243894), octamer-binding transcription factor 4 (Oct-4; Abcam; 1:10,000; cat. no. ab200834), B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1; Abcam; 1:10,000; cat. no. ab126783), inhibitor of nuclear factor κ B α (IκBα; Abcam; 1:1,000; cat. no. ab76429), phosphorylated (p)-IκBα (Abcam; 1:10,000; cat. no. ab133462), p65 (Abcam; 1:5,000; cat. no. ab32536), p-p65 (Abcam; 1:1,000; cat. no. ab76302), JNK (Cell Signaling Technology, Inc; 1:1,000; cat. no. 9252), p-JNK (Cell Signaling Technology, Inc. 1:1,000; cat. no. 9251), p38 (Cell Signaling Technology, Inc.; 1:1,000; cat. no. 212), p-p38 (Cell Signaling Technology, Inc; 1:1,000; cat. no. 9216), MEK (Cell Signaling Technology, Inc.; 1:1,000; cat. no. 4694), p-MEK (Cell Signaling Technology, Inc.; 1:1,000; cat. no. 9154) and GAPDH (Abcam; 1:5,000; cat. no. ab8245). GAPDH served as the loading control. ImageJ software (v1.5; National Institutes of Health) was used for densitometry analysis.
All experimental procedures performed in the present study were approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College (Chengdu, China). A total of 30 male 6-week-old nude mice (weight, 18–22 g) were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. Mice were maintained in pathogen-free devices under a controlled temperature (24±1°C) and 55% humidity with a 12-h light/dark cycle. Mice were raised with free access to food and water. After adaption for 1 week, mice were subcutaneously injected with 100 µl NCI-H460-CSC cells (2.5×106 cells), which were resuspended in ice-cold PBS. Subsequently, mice (n=10 in each group) were randomly assigned to three groups, including control, 400 and 800 mg/kg SchB groups. Mice in the control group were administrated with PBS by oral gavage, while mice in the other two treatment groups were administered with 400 or 800 mg/kg SchB by oral gavage every day for 5 weeks. The size of tumors was measured at 1, 3, 7, 11, 14, 21, 28 and 35 days after cancer stem-like cell injection, and xenograft volume was calculated with the following formula: Volume = ½ (L × W2). At the end of the experiment, neoplasms were removed and weighed after all mice were euthanized by CO2 asphyxiation, at a flow rate of 30% volume displaced/minute (
Tumor tissues were fixed by 4% paraformaldehyde at 4°C for 72 h, and embedded in paraffin. Paraffin-embedded tumor samples were sliced into 5-µm-thick slides and subjected to heat pretreatment at 60°C for 1 h, followed by deparaffinization, and then rehydrated with xylene and a graded ethanol series. Following quenching using 3% hydrogen peroxide aqueous solution, sections were blocked with 10% goat serum (EMD Millipore) for 1 h at room temperature. Sections were incubated with a primary antibody for proliferating cell nuclear antigen (PCNA; Abcam; 1:500; cat. no. ab92552) at 4°C overnight, probed with HRP-conjugated secondary antibody (Abcam; 1:1,000; cat. no. ab6721) at 37°C for 30 min, treated with 3,3-diaminobenzidine and finally stained with hematoxylin at room temperature for 5 min. Sections were photographed under an inverted light microscope (Nikon Corporation; magnification, ×100).
Cancer stem-like cells (5×104 cells) were seeded on glass slides, immobilized in 4% formaldehyde at 4°C for 15 min, followed by permeation with 0.3% Triton X-100 and blocked using 10% goat serum (EMD Millipore) at room temperature for 30 min. Subsequently, slides were probed using an anti-p65 antibody (Abcam; 1:100; cat. no. ab32536) overnight at 4°C, incubated with rhodamine-labeled secondary antibody (Invitrogen; Thermo Fisher Scientific, Inc.; 1:350; cat. no. 31670) at room temperature for 1 h and stained with DAPI at room temperature for 5 min in the dark. Subsequently, the fluorescence signals were observed with a fluorescence microscope (BX61; Olympus Corporation; magnification, ×400).
All statistical analyses were performed using SPSS 16 software (SPSS, Inc.). Results were presented as the mean ± SD from three independent assays. Differences between 2 groups were analyzed by unpaired Student's t-test and differences among >2 groups were analyzed by one-way or two-way ANOVA with Tukey's correction. P<0.05 was considered to indicate a statistically significant difference.
To establish cancer stem-like cells, NCI-H460 and H661 cells were treated with serum-free DMEM/F12 medium containing the aforementioned growth factors. Considering that CD133+ cells exhibit stemness features, flow cytometry analysis was used for evaluation of CD133+ cells (
Subsequently, the effects of SchB on migration and invasion of cancer stem-like cells were investigated. Due to the cytotoxicity of SchB at concentrations ≥40 µmol/l, cancer stem-like cells were treated with 10, 20 or 40 µmol/l SchB for subsequent experiments. The wound healing assay revealed that the migratory ability of NCI-H460-CSCs was significantly decreased by 10–40 µmol/l SchB treatment compared with the control group (P<0.05 and P<0.01), while the migratory ability of H661-CSCs was significantly decreased by 20–40 µmol/l SchB treatment compared with the control group (P<0.01;
Thereafter, the present study intended to assess the potential of SchB in CSC characteristics of NCI-H460-CSCs and H661-CSCs. As shown in
In order to investigate the mechanism underlying SchB activity, the impact of SchB on key signaling pathways involved in tumor progression was assessed. The present results indicated that SchB significantly decreased the phosphorylation levels of IκBα (P<0.01) and p65 (P<0.05 and P<0.01 for NCI-H460-CSCs, and P<0.01 for H661-CSCs) compared with the control group (
To confirm the role of the NF-κB and p38 MAPK signaling pathways in the tumor-suppressive properties of SchB, rescue experiments were performed. Considering the similar effects of SchB on NCI-H460-CSCs and H661-CSCs, rescue experiments were validated in NCI-H460-CSCs. The NF-κB and p38 MAPK signaling pathways were activated via treatment with TNF-α and anisomycin, respectively. As demonstrated in
Based on the aforementioned results, the present study aimed to verify whether SchB had a tumor suppressor function in large-cell lung cancer
The present study focused on the potentially tumor-suppressive role of SchB in large-cell lung cancer. NCI-H460 and H661 cells were used as protocells to induce CSCs. Notably, it was revealed that SchB suppressed migration, invasion, EMT and stemness in CSCs. The NF-κB and p38 MAPK signaling pathways were inactivated in SchB-treated cells, and effects of activating the NF-κB or p38 MAPK signaling pathways were abolished by SchB. Effects of SchB on CSCs were also confirmed
Mounting evidence has illustrated that CSCs serve an important part in the occurrence and development of tumors on account of their ability for self-renewal, differentiation and induction of tumor growth (
The present findings revealed that SchB led to toxicity in both cancer stem-like cells and protocells at concentrations ≥40 µmol/l. Cells were therefore treated with 10, 20 or 40 µmol/l SchB in subsequent experiments to avert potential toxic effects of SchB on migration, invasion, EMT and stemness. Increasing studies have demonstrated that cell migration and EMT are associated with the features of cancer stem-like cells (
As a transcription factor, NF-κB is involved in regulating the expression levels of numerous crucial genes associated with apoptosis, inflammation, tumorigenesis and the progression of autoimmune diseases (
In order to certify whether the functional role of SchB in large-cell lung cancer cells was mediated by the NF-κB and p38 MAPK signaling pathways, the activation of these pathways was induced using the corresponding pathway activators (TNF-α and anisomycin, respectively) and rescue experiments were performed. It was demonstrated that SchB abrogated the effects of NF-κB and p38 MAPK activation on large-cell lung cancer cell migration and invasion. Moreover, EMT and stemness features restrained by SchB were retrieved when NCI-H460-CSC cells were treated with TNF-α or anisomycin, further expounding the antitumor activities of SchB in large-cell lung cancer cells through repression of the NF-κB and p38 MAPK signaling pathways. In agreement with these results, animal assays indicated that SchB served as a tumor suppressor in the malignant phenotype of large-cell lung cancer
To the best of our knowledge, the present study was the first to clarify the role and molecular mechanism of SchB in EMT and stemness traits of large-cell lung cancer cells. It was demonstrated that SchB suppressed cell migration, invasion, EMT and CSC properties in large-cell lung cancer via regulation of the NF-κB and p38 MAPK signaling pathways. However, the specific regulatory mechanism underlying SchB in the inactivation of the NF-κB and p38 MAPK signaling pathways, and the association between these pathways remain unclear. Additionally, other EMT-associated signaling pathways, such as SMAD, PI3K/AKT, Wnt and Notch, may also participate in the SchB-involved regulation, and should be further investigated in future studies. The potential difference of tumorigenesis between cancer stem-like cells and parental cells is also a topic that requires further exploration. Overall, the present findings confirmed the anticancer function of SchB in cancer stem-like cells, revealing that SchB may be used as a promising drug for the treatment of large-cell lung cancer.
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
HW, LW and RM designed the study. SL completed all the assays. SL and LW assessed all the raw data to ensure its legitimacy, and conducted data analyses. SL and LW were responsible for writing the manuscript. All authors read and approved the final manuscript, and are responsible for confirming the authenticity of the data.
All experimental animal procedures performed in the present study were approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College (Chengdu, China; approval no. SCXK 2018-19).
Not applicable.
The authors declare that they have no competing interests.
Cell Counting Kit-8
cancer stem cell
epithelial-mesenchymal transition
radioimmunoprecipitation
immunohistochemical
Schisandrin B
SchB leads to toxicity in cancer stem-like cells. To induce cancer stem-like cells, NCI-H460 and H661 cells were incubated in serum-free DMEM/F12 medium containing appropriate growth factors. (A) Following induction, the ratio of CD133+ cells was analyzed by flow cytometry. Blue cells represent CD133− cells and red cells represent CD133+ cells. (B) Expression levels of CSC markers (CD44, Oct-4 and Bmi-1) were detected by western blot analysis. (C) Sphere-forming assay was performed to estimate CSC traits of large-cell lung cancer cells. Scale bar, 25 µm. **P<0.01 vs. parental cells. (D) Cell cycle was evaluated by flow cytometry. **P<0.01 G0/G1 parental cells vs. cancer stem-like cells. (E) Chemical structure of SchB. (F) Cancer stem-like cells and parental cells were incubated with different doses of SchB (0, 5, 10, 20, 40, 80, 120 and 160 µmol/l) for 72 h and Cell Counting Kit-8 assay was utilized to examine the viability of cancer stem-like cells and parental cells. Cells treated with 0 µmol/l SchB acted as control. Differences were analyzed via one-way ANOVA with Tukey's correction. *P<0.05 and **P<0.01 vs. 0 µmol/l. All experimental data are shown as the mean ± SD (n=3). SSC, side scatter; SchB, Schisandrin B; CSC, cancer stem cell; Oct-4, octamer-binding transcription factor 4; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog.
SchB suppresses migration, invasion and EMT of cancer stem-like cells. Cancer stem-like cells (NCI-H460-CSCs and H661-CSCs) were treated with 10, 20 or 40 µmol/l SchB and the control group was treated with PBS. (A) Migration was detected using wound healing assays. Scale bar for NCI-H460-CSC, 50 µm; Scale bar for H661-CSC, 100 µm. (B) Transwell assays were performed to assess cell invasion in cancer stem-like cells. Scale bar for NCI-H460-CSC, 50 µm; Scale bar for H661-CSC, 100 µm. (C) Protein expression levels of EMT markers including E-cadherin, vimentin and N-cadherin were determined by western blot analysis. All experimental data are shown as the mean ± SD (n=3). Differences were analyzed via one-way ANOVA with Tukey's correction. *P<0.05, **P<0.01 vs. control. SchB, Schisandrin B; EMT, epithelial-mesenchymal transition.
SchB alleviates the stemness of cancer stem-like cells. Cancer stem-like cells (NCI-H460-CSCs and H661-CSCs) were treated with 10, 20 or 40 µmol/l SchB and the control group was treated with PBS. (A) Flow cytometry was used to evaluate the number of CD133+ cells. (B) CD133, CD44, Oct-4 and Bmi-1 expression was tested by western blot analysis. (C) Sphere-forming ability of cancer stem-like cells was estimated by sphere-forming assay. Scale bar, 25 µm. All experimental data are shown as the mean ± SD (n=3). Differences were analyzed by one-way ANOVA with Tukey's correction. *P<0.05, **P<0.01 vs. control. SSC, side scatter; SchB, Schisandrin B; Oct-4, octamer-binding transcription factor 4; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog.
SchB results in the inhibition of the NF-κB and p38 MAPK signaling pathways. Cancer stem-like cells (NCI-H460-CSCs and H661-CSCs) were treated with 10, 20 or 40 µmol/l SchB and the control group was treated with PBS. (A) Protein expression levels of important genes in the NF-κB signaling pathway (IκBα and p65) were examined by western blot analysis. (B) Immunofluorescence assay was used to determine the nuclear translocation of p65 in cancer stem-like cells after SchB treatment. Scale bar, 25 µm. (C) Protein expression levels of JNK, p-JNK, p38, p-p38, MEK and p-MEK were measured by western blot analysis. Differences were analyzed by one-way ANOVA with Tukey's correction. *P<0.05, **P<0.01 vs. control. SchB, Schisandrin B; IκBα, inhibitor of nuclear factor κ Bα; p, phosphorylated.
SchB attenuates the CSC properties of NCI-H460-CSCs via regulation of the NF-κB and p38 MAPK signaling pathways. To explore the role of the NF-κB and p38 MAPK signaling pathways in the effects of SchB on large-cell lung cancer, NCI-H460-CSCs were treated with 40 µmol/l SchB for 12 h and then stimulated with 10 ng/ml TNF-α or 2 ng/ml anisomycin for 30 min. Cells administrated with PBS or SchB alone were used as controls. Western blot was implemented to detect the expression levels of associated proteins in the (A) NF-κB and (B) MAPK signaling pathways. Differences were analyzed via two-way ANOVA with Tukey's correction. **P<0.01 vs. control. ##P<0.01 vs. SchB. &&P<0.01 TNF-α/anisomycin. (C) Cell migration and (D) invasion were assessed by wound healing and Transwell assays, respectively. Scale bar, 50 µm. (E) Expression levels of EMT-associated proteins were examined by western blot analysis. (F) Proportion of CD133+ cells was analyzed by flow cytometry. Blue cells represent CD133− cells and red cells represent CD133+ cells. (G) Protein expression levels of CD133, CD44, Oct-4 and Bmi-1 were determined by western blot analysis. (H) Sphere-forming assay was used to evaluate stemness in NCI-H460-CSCs. Scale bar, 25 µm. All experimental data are shown as the mean ± SD (n=3). **P<0.01. SSC, side scatter; SchB, Schisandrin B; CSC, cancer stem cell; Oct-4, octamer-binding transcription factor 4; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog; IκBα, inhibitor of nuclear factor κB α; p, phosphorylated.
SchB restrains the growth and CSC characteristics of NCI-H460-CSCs