Glioblastoma (GBM) is a highly lethal brain tumor that is resistant to most therapeutic means currently available. It accounts for 70% of human primary malignant brain tumors [1
]. The current treatment for GBM patients mostly depends on surgical resection in combination with radiotherapy and chemotherapy [2
]. Despite significant advances in cancer treatment, little improvement has been achieved in the prognosis of GBM patients over decades [3
Cancer stem cells represent a subpopulation of tumor cells with enhanced clonogenic and tumorigenic potentials within a tumor [4
]. A variety of tumors have been reported to contain a cell population with stem cell-like properties, including self-renewal and differentiation [5
]. A number of tumor types, including gliomas, have shown the existence of a unique population of tumor/glioma initiating cells [4
], which were initially isolated from human gliomas for isolating neural stem cells. These are biologically characterized as embryonic and/or tissue-restricted stem cells with self-renewal capacity and ability to differentiate along tissue-specific lineage [6
]. Such stem cells are believed to cause relapse after tumor resection, thereby contributing to treatment resistance [7
]. Glioma stem cells can be enriched by cell sorting via cell surface proteins such as CD44 and CD133 [8
] and are thus amenable to investigations focusing on factors that influence their functions.
Among the molecules contributing to tumorigenesis, epidermal growth factor receptor (EGFR) has been recognized to induce neoplastic transformation in the presence of ligands and its overexpression is characteristic of several cancer cell lines [9
]. Specifically, in human gliomas EGFR is not only overexpressed but also occurs in several mutations [10
]. It is activated by mitogenic ligands, including epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphiregulin, epiregulin and betacellulin [11
]. Ligand binding leads to homo- or heterodimerization of EGFR with other ligand-bound ErbB receptors, thus transmitting mitogenic signals to downstream cell survival and proliferation involved signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) [9
Honokiol, used in traditional Chinese medicine, is a natural compound extracted from the bark of the magnolia tree. Studies have demonstrated a wide range of its pharmaceutical effects, including anti-inflammatory, anti-microbial and antioxidant activities as well as protection against hepatotoxicity, neurotoxicity, thrombosis and angiopathy [12
]. Honokiol has also been reported to target EGFR signaling via STAT3 in the treatment of head/neck cancer, thus enhancing the effects of EGFR-targeting therapies [13
]. It affects tumor cell growth and survival by inhibition of the nuclear factor kappa B (NFκB) signaling pathway and altering other molecular targets in several cancer models [14
Here, we show that honokiol inhibits glioma cell viability and migration/invasion and induces apoptosis of human glioma/glioblastoma cell lines. We also show that honokiol reduces EGFR expression and down-regulates JAK/STAT3 signaling pathways. In addition, honokiol decreases the number of CD133 positive stem cells and promotes elimination of a glioma stem cell subpopulation. In vivo, honokiol profoundly suppresses glioblastoma xenograft growth in nude mice and in zebrafish yolk sack.
Glioblastoma accounts for 46.6% of malignant central nervous system tumors in adults [15
]. Despite advanced surgery and radio-/chemotherapy developed for treatment of gliomas, the median overall survival is about 14 months [16
], with a 5-year survival rate of only 5.5% [15
]. In search for an agent that may enhance patient survival, we here show that honokiol ameliorates several indicators of glioma/glioblastoma malignancy, such as migration, invasion, viability and colony formation of stem cells. These findings suggest that honokiol may exert its anticancer functions by inhibition of these parameters via intricate signaling as evidenced in the present study.
Apoptotic signaling, inhibiting tumor progression, promotes cellular self-destruction [17
]. Anti-apoptotic proteins promote development of many malignant tumors, especially of gliomas [19
]. Up-regulation of anti-apoptotic Bcl-2 and Bcl-XL expression contributes to glioblastoma recurrence, in parallel with down-regulation of the pro-apoptotic protein Bax [20
]. Down-regulation of Bcl-2 or Bcl-XL results in glioma cell death and sensitization to chemotherapy and radiotherapy [21
]. Honokiol induces apoptosis by enhancing p53 and reducing anti-apoptotic gene expression in human colorectal cancer cells [24
An important mediator of tumor progression is EGFR, whose expression correlates positively with malignancy and contributes to poor prognosis [25
]. EGFR is involved in Jak/STAT, nfb, mTOR and MAPK signaling pathways, thereby influencing cancer cell self-renewal, proliferation, differentiation and migration [26
]. Honokiol can induce mitochondria-dependent and death receptor-mediated apoptosis in multi-drug resistant KB cells by inhibition of EGFR-STAT3 signaling and down-regulation of STAT3 target genes [29
]. Honokiol has also been identified as a small molecule agonist of cell adhesion molecule L1 [30
]. A L1 fragment (L1-70) is imported from the cell surface via the cytoplasm into mitochondria and increases ATP production by specifically binding to the NDUFV2 enzyme in complex 1 [31
]. It remains to be seen how this anti-apoptopic effect of honokiol influences gliomas/glioblastomas. Since the effect of honokiol is highly dose-dependent and was not systematically evaluated in mitochondrial metabolism, it is likely that honokiol, at a high dose, may interfere with this cleavage process, thus partially contributing to mitochondrial malfunction and thereby apoptosis.
Cancer stem cells are the most vital sub-population of cancer cells contributing to drug-resistance and tumor recurrence. Glioma/glioblastoma cells contain a sub-population of glioma initiating cells, which may contribute to chemotherapy-resistance. Previous reports demonstrated that honokiol suppressed metastasis of renal cancer cells via blocking both epithelial-mesenchymal transition and cancer stem cell phenotypes [24
]. Honokiol potently inhibits melanoma cell metastasis, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling [32
]. In agreement with these reports, we found that honokiol reduces levels of EGFR, CD133 and Nestin, indicating that it may attenuate the stemness of glioma stem cells by inhibiting EGFR expression. Thus, a combined application of honokiol and other therapeutic means targeting tumor stem cells may prove effective in treating high-grade glioma.
MMPs are a large family of zinc-dependent endopeptidases that are capable of cleaving multiple extracellular matrix proteins. MMPs can be subdivided into multiple categories depending on their substrate specificity, such as collagenases, stromelysins and gelatinases [33
]. Invasive gliomas show MMP2 and MMP9 overexpression and both MMP2 and MMP9 play important roles in infiltrative growth of gliomas [34
]. In addition, E-cadherin repression could enhance the generation of cancer stem cells [35
]. Increased E-cadherin expression may thus contribute to the attenuation of cancer development. It was also reported that NF-κB promotes migration and invasion of cholangiocarcinoma cells by up-regulating Snail and consequent repression of E-cadherin [36
]. We found that honokiol inhibited NF-κB expression, while increasing that of E-cadherin, suggesting that honokiol may inhibit migration and invasion of glioma cells in part by inhibition of MMP2/9 expression, as well as attenuation of the NF-κB-mediated E-cadherin pathway.
The Ras/MAPK/Erk and PI3K/Akt signaling pathways exert crucial roles in cell growth, differentiation, survival and apoptosis in many cancer types [37
]. Honokiol has been shown to inhibit cell proliferation by suppression of the Erk signaling pathway in neuroblastoma cancer cells [43
]. Thus, our data imply that honokiol may attenuate proliferation and promote cell apoptosis by inhibition of the PI3K/Akt and MAPK/Erk signaling pathways in glioma cells, which may further inhibit cell migration and colony formation.
To extend these in vitro observations, we performed in vivo experiments to study the effects of honokiol on the growth of glioblastoma U-87 MG xenografts in a zebrafish model [44
] and in nude mice. We show that honokiol remarkably suppresses glioblastoma growth in xenografts, in that more apoptotic cells were observed in honokiol-treated glioma tissue. Moreover, EGFR, phosphorylated STAT3, CD133 and Nestin expression was reduced in xenografted glioma tissues in response to honokiol treatment, which was consistent with in vitro results we observed. Honokiol exhibited a profoundly effective inhibition of glioma growth in vivo as observed in the present study. Altogether these results indicate that honokiol inhibits glioma growth and invasion mainly by inhibiting proliferation of glioma stem cells. Unlike intracerebral orthotropic implantation, there is no formation of blood brain barrier (BBB) in the subcutaneous glioma xenograft. This may enhance drug utilization and therapeutic efficacy of honokiol. Intravenous administration of honokiol can effectively attenuate spinal cord lesions in a mouse spinal cord injury model [42
], indicating that honokiol may access targeted glioma cells through BBB penetration.
An early report demonstrated that honokiol exerts a preventively anticancer effect by inhibiting metastasis and inducing apoptotic cell death of brain tumor cells [45
]. Honokiol dose-dependently inhibited the proliferation of stem-like side population (SP) cells of GBM and strongly reversed Temozolomide (TMZ) resistance by inducing apoptosis in GBM8401 cells [46
]. It is recently implicated that honokiol can enhance TMZ-induced apoptotic insults to glioma cells via an intrinsic mitochondrion-dependent mechanism [47
]. Our results advanced these reports by demonstrating that honokiol may exert these auxiliary anti-cancer roles via an EGFR-mediated JAK-STAT3 signaling pathway. It remains elucidation whether honokiol can function like N6-isopentenyladenosine (iPA), which has been reported to display an immune-mediated antitumor activity [48
In summary, with the combined application of both in vitro and vi vivo experiments, we advanced these reports by further clarifying that he traditional Chinese herb honokiol can inhibit glioma stem cell proliferation by inducing the apoptosis of glioma stem cells via EGFR-mediated JAK-STAT3 signaling pathway. Honokiol contributes to EMT inhibition by increasing the expression of E-Cadherin. Honokiol also Inhibits glioma/glioblastoma progression by targeting EGFR. The combined observations indicate that honokiol may represent a novel therapy for malignant glioma.
4. Materials and Methods
4.1. Cells and Reagents
U251 human glioma and U-87 MG human glioblastoma cell lines were purchased from the Chinese Type Culture Collection (CTCC, Shanghai, China) and were maintained in Dulbecco’s modified Eagle’s medium low Glucose (DMEM, SH30021.01, Thermo Scientific HyClone, Beijing, China) supplemented with 50 U/mL of a penicillin/streptomycin mixture (Solarbio Biotech, Beijing, China) and 10% fetal bovine serum (Sijiqing Biotech, Hangzhou, China). CD133+ cells were maintained in DMEM-F12 medium supplemented with 20 ng/ml of recombinant human epidermal growth factor (EGF) (10605-HNAE) and recombinant human basic fibroblast growth factor (bFGF) (10014-HNAE) and 2% B27 supplement (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (Solarbio Biotech). The cells were routinely grown in 60-cm2 cell culture plates (Corning Incorporated, Corning, NY, USA) at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO2). Honokiol and Stattic were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MTT and TUNEL assay kits were purchased from Beyotime Biotechnology (Haimen, Jiangsu, China).
4.2. MTT Assay
U251 and U-87 MG cells were seeded into a 96-well plate at a density of 3 × 103 cells per well. After overnight incubation, the culture medium was aspirated and honokiol’s effect on cell viability cells was measured at doses ranging from 0 to 60 μM in complete culture medium after incubation times of 24 and 48 h in 0.1% DMSO, with a vehicle control also comprising 0.1% DMSO. Then, 10 µl of MTT (5 mg/ml; Beyotime) was added to each well and cells were further cultured for 4 h. After removal of the culture medium, 100 µl of DMSO was added. The absorbance was measured at a wavelength of 490 nm in an ELISA plate reader (Infinite M1000, Tecan, Switzerland). Cell survival was determined using the formula: Survival rate (%) = mean ODtreated groups/ODvehicle control group. The half-maximal inhibitory concentration (IC50) at 48 h was calculated, with the survival of vehicle-treated cells being set at 100%. Results are from three independent experiments.
4.3. Scratch Assay
U-87 MG cells were seeded at a density of 5 × 104 cells per well into a 96-well plate in complete cell culture medium until confluency. After treatment with different concentrations of honokiol, the monolayer was scratched with a 10 μL plastic pipette tip to create a space. The space width was then examined under a phase-contrast microscope at × 100 magnification (Olympus IX51, Tokyo, Japan) after 0, 24 and 48 h of incubation. Photographs of at least three random fields were taken and cell spreading into the space was determined by measuring the gap width. Results are from at least three independent experiments.
4.4. Invasion Assay
For this assay, U251 and U-87 MG cells were incubated with 0, 20, 40, 60 μM honokiol for 48 h. Then, 1 × 104 cells in 100 μL medium without fetal bovine serum were seeded onto a polycarbonate membrane insert with 8 μm pores (underside precoated with fibronectin) in a Transwell Apparatus (Costar, Corning) and seeded onto the upper side of the membrane pre-coated with Matrigel (Becton Dickinson Biosciences, Palo Alto, CA, USA). The lower chamber was loaded with 500 μL medium supplemented with 10% fetal bovine serum. After incubation for 12 h at 37 °C in a 5% CO2 atmosphere, the membrane was washed with PBS and cells on the membrane top surface were removed with a cotton swab. Cells adhering to the underside were fixed with 4% formaldehyde, stained with crystal violet solution and counted under a microscope. Results are from three independent experiments.
4.5. Colony Formation Assay
U251 and U-87 MG cells (1500 cells/well) were seeded into a 24-well plate. After treatment with 0, 20, 40, 60 μM honokiol at 37 °C for 10 days, the colonies were fixed with methanol for 20 min, stained with 0.1% crystal violet and visualized under a phase-contrast light microscope (Olympus IX51). A cluster of more than 50 cells was identified as a colony. Results are from three independent experiments.
4.6. Flow Cytometric Assay of Apoptosis
U251 and U-87 MG cells were seeded at a density of 5 × 105 cells per well into 6-well plates in complete culture medium. After overnight incubation, the culture medium was removed and the cells were treated with honokiol at doses ranging from 0 to 60 µM in complete culture medium. Cells were then further cultured for 24 h and 48 h. Apoptosis was evaluated with the Annexin V-EGFP/PI Apoptosis Kit according to the manufacturer’s protocol (Beyotime) using the Accuri C6 flow Cytometer (Becton Dickson Biosciences). Annexin V-EGFP/PI was excited with 495 and 535 nm lasers and signals were detected using 530/30 and 585/40 band pass filters, respectively.
4.7. Assay for CD133-Positive Cells
U-87 MG cells were seeded at a density of 5 × 105
cells per well into 6-well plates, incubated with 0 to 60 µM honokiol in DMEM-F12 medium supplemented with 20 ng/ml of recombinant human EGF (Sigma-Aldrich, St. Louis, MO, USA) and recombinant human bFGF (Sigma) and 2% B27 supplement (Invitrogen) and 1% penicillin/streptomycin solution (Solarbio Biotech). After 7 days, cultures were evaluated for CD133 immunopositive stem cells by flow cytometry (see Section 4.8
4.8. Flow Cytometric Cell Sorting
U251 and U-87 MG cells were trypsinized and washed with PBS three times for 3 min each, followed by a wash in DMEM-F12 and FACS buffer (PBS containing 1% BSA). Then, 107 cells were resuspended in 2 ml FACS buffer on ice and 10 μL of anti-CD133-PE antiserum (Miltenyi, Cat. No. 130-090-853; Bergisch Gladbach, Germany) was added. Cells were then incubated on ice for 15 min in the dark, pelleted by centrifugation at 1000× g for 5 min at 4 °C and washed once with FACS buffer. Stained cells were sorted with the FACSII (Becton and Dickinson Biosciences). Cell debris were gated out using a forward scatter/sideward scatter dot plot. CD133-negative and CD133-positive cell populations were collected.
4.9. Flow Cytometry of CD133+ Cells
U251 and U-87 MG CD133+ cells were cultured at a density of 5 × 105 cells per well into 6-well plates in DMEM-F12 medium supplemented with 20 ng/ml of each recombinant human EGF (Sigma) and recombinant human bFGF (Sigma) and 2% B27 supplement (Invitrogen) and 1% penicillin/streptomycin solution (Solarbio Biotech). Effects of honokiol or Stattic, as well as their combined effects on the stemness of CD133+ cells were evaluated by treatment with 60 µM honokiol, 20 µM Stattic and mixture of 60 µM honokiol and 20 µM Stattic in DMEM-F12. Cells were then further cultured for 48 h. Thereafter cells were washed with PBS three times for 3 min each and washed in DMEM-F12 and FACS buffer (PBS containing 1% BSA). 107 cells were resuspended in 2 ml FACS buffer on ice. Ten µl of anti-CD133-PE antiserum was added and cells were incubated for 15 min on ice in the dark. Cells were pelleted and washed once with FACS buffer. Stained cells were measured using the Accuri C6 Cytometer (Becton Dickinson Biosciences).
4.10. Immunofluorescence Staining
U251 and U-87 MG cells were seeded into glass coverslip in a 24-well plate at a density of 5 × 104 cells per well. After overnight incubation, the culture medium was removed and the cells were incubated with honokiol at doses ranging from 0 to 60 µM in complete culture medium and cells were further cultured for 48 h. Fixed U251 and U-87 MG cells were incubated with anti-EGFR (ErbB1) antibody (1:200; Cat. No. 66455-1-Ig, Proteintech, Rosemont, IL, USA) overnight at 4 °C. After washing in PBS three times for 5 min each, the samples were incubated at room temperature with FITC-conjugated goat anti-mouse IgG antibody (1:500; Jackson ImmunoResearch, West Grove, PA, USA) for 60 min at room temperature. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted using anti-fade mounting solution (Beyotime).
4.11. Immunofluorescence Staining of CD133+ Cells
CD133+ cells were seeded onto glass coverslips coated with poly-L-lysine (Sigma-Aldrich). After 12 h they were incubated with honokiol or Stattic at doses of 0, 20 and 60 µM for 48 h in DMEM/F12 culture medium. Cells were fixed and incubated with antibodies against CD133/1 (1:50; Cat. No. 18470-1-AP, Proteintech) and Nestin (1:50; Cat. No. 66259-1-Ig, Proteintech) overnight at 4 °C After washing in PBS three times for 5 min each, the samples were incubated at room temperature with goat anti-mouse fluorescein (FITC)–conjugated antibody (1:500; Jackson ImmunoResearch) and anti-rabbit secondary antibody conjugated to DylightTM 594 (1:500; Jackson ImmunoResearch) for 60 min. The cells were then stained with DAPI and mounted using anti-fade mounting solution (Beyotime).
4.12. Western Blot Analysis
Cell lysates were heated at 100 °C in sample loading buffer (0.125 mol/L Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 0.1% bromophenol blue and 5% β-mercaptoethanol), resolved by 10% SDS polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (PVDF, Merck KGaA, Darmstadt, Germany). Non-specific protein binding sites were blocked with 5% BSA (Cat. No. #A8020, Solarbio, Beijing, China) diluted in Tris-buffered saline buffer containing 0.05% Tween-20 (TBST, pH 7.4). The membranes were incubated with antibodies specific for ERK (MK1; 1:1000; Cat. No. sc-135900, this and the following antibodies were purchased from Santa Cruz Biotechnology and the dilutions and catalog numbers are indicated for each antibody), AKT (G-5; 1:1000; Cat. No. sc-53373), p-ERK (E-4; 1:1000; Cat. No. sc-7383), p-AKT (11E6; 1:1000; Cat. No. sc-81433), Bcl-2 (C-2; 1:1000; Cat. No. sc-7382), Bax (C-19; 1:1000; Cat. No. sc-526), STAT3 (H-190; 1:1000; Cat. No. sc-7179), p-STAT3 (Ser 727; 1:1000; Cat. No. sc-8001-R), NFkB (A; 1:1000; Cat. No. sc-109), MMP9 (C-20; 1:1000; Cat. No. sc-6840), MMP2 (K-20; 1:1000; Cat. No. sc-8835), beta-catenin (6F9; 1:1000; Cat. No. sc-53484), E-cadherin (H-108; 1:1000; Cat. No. sc-7870) and GAPDH (2E3-2E10; 1:1000; Cat. No. sc-293335). In the following used antibodies are both against pro- and cleaved caspase-3 (1:500; Cat. No. BM3257, Boster; Wuhan, China), CD133 (1:1000; Cat. No. 18470-1-AP, Proteintech); Nestin (1:1000; Cat. No. 66259-1-Ig, Proteintech); EGFR (1:1000; Cat. No. 66455-1-Ig, Proteintech). They were also incubated overnight at 4 °C. After 3 washes for 5 min each, HRP-conjugated goat anti-mouse (Cat. No. BA1051, Boster) and goat anti-rabbit (Cat. No. BA1055, Boster) secondary antibodies (1:1000 for both; Boster) diluted in 5% BSA in TBST were applied, followed by 3 washes with TBST for 5 min each at room temperature. Signal intensities were quantified using Image J software (https://imagej.nih.gov/ij/
) as the average densitometry multiplied by the area (measured as the number of pixels).
4.13. Zebrafish Xenografting
Zebrafish were maintained according to standard procedures [49
]. Wild type zebrafish were purchased from Huiyuan Aquatic Animals Company (Shantou, Guangdong, China). Twenty-four h post fertilization (dpf), larvae were treated with PTU (1-phenyl 2-thiourea, 0.2 mM) to remain transparent for microscopic analysis. Two dpf larvae were anaesthetized with 0.003% tricaine (Sigma) and positioned on a 10 cm dish coated with 3% agarose. Cell suspensions were injected by a micromanipulator (LPP01-100, LongerPump, Wuhan, China) equipped with borosilicate glass capillary needles (1.5 mm in outer diameter and 0.86 mm in inner diameter) which were made by a P-97 micropipette puller (Sutter Instrument Company, Novato, CA, USA). Approximately 200 cells in a volume of 10 μL were injected into the yolk sac of larvae, which were then maintained in E3 medium/PTU for 1 h at 28 °C and 32 °C. Images were captured at 0- and 3-day time points post injection using a Zeiss fluorescence microscope (Axio Observer A1, Oberkochen, Germany). All experiments were approved by the local authorities (ethical code: SUMC2018-125, permission date: 30 June 2018).
4.14. Nude Mouse Xenografting
Four-week-old nude female mice were purchased from the Model Animal Research Center of Nanjing University and maintained at the animal facility of Shantou University Medical College for 1 week prior to experimental use. U-87 MG cells (5 × 105 cells/mouse) were subcutaneously injected into the right flank near the upper extremity. After the tumors had reached a volume of 2 mm3, mice were randomized into two groups (n = 3 per group). Then, either honokiol (100 mg/kg/day) or DMSO vehicle control were intraperitoneally injected daily for 7 days. The tumor size was measured using a Vernier caliper (BST-01601, BESTIR, Foshan, Guangdong, China) at days 7, 10, 13, 16, 18 and 21 after treatment. Tumor volume was estimated by the formula (L × S2/2, L: largest diameter and S: smallest diameter). Mice were sacrificed and tumors were dissected out and weighed using an electronic balance (ME104, Mettler Toledo, Shanghai, China). All experiments were approved by the Institutional Animal Care and Use Committee of Shantou University Medical College (ethical code: SUMC2018-119, permission date: 30 May 2018).
4.15. H & E Staining and Immunofluorescence Staining of Xenografted Tumor Cells
Tumors were cryosectioned at 4 μm for hematoxylin/eosin staining as described [50
]. For estimation of apoptosis, xenograft tumor tissues were evaluated by both DAPI staining and terminal deoxynucleotidyl transferase (TdT) deoxyuridine 5-triphosphate dUTP Nick-End Labeling (TUNEL) staining (Beyotime). Sections were washed using PBS and incubated with TF3-dUTP and DAPI for 60 min at 37 °C. Both TUNEL-positive and Cy3 (Cyanine 3)-positive cells were inspected with a fluorescence microscope (Axio Imager A2, Oberkochen, Germany). Photographs of at least five random fluorescent fields were taken.
Immunohistochemical staining was performed for evaluation of expression and localization of EGFR, pSTAT3, CD133 and Nestin. In brief, deparaffinized sections were rehydrated through a graded series of ethanol to phosphate buffered saline (PBS). Antigen retrieval was performed and endogenous peroxidase clearance was performed by incubation with 3% H2O2. Sections were then blocked with 10% normal goat serum (AR009, Boster) for 30 minutes and individually incubated with rabbit anti-CD133 (1:200; Cat. No. 18470-1-AP, Proteintech), mouse anti-Nestin (1:200; 66259-1-Ig, Proteintech), mouse anti-EGFR (1:1000; 66455-1-Ig, Proteintech), rabbit anti-p-STAT3 (1:200; Cat. No. sc-8001-R, Santa Cruz) antibodies. The antigen-antibody complexes were visualized using the AEC method (Zhongshan Goldbridge Biotechnology, Beijing, China) and counterstained with hematoxylin (Zhongshan Goldbridge Biotechnology). Immunohistochemical staining was analyzed using a Jiangnan light microscope DN-10B (Jiangnan, Nanjing, Jiangsu, China).
Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) 19.0 software (SPSS, Chicago, IL, USA). The intensities were quantified using Image J software. Data are expressed as means ± SEM of at least 3 to 4 independent experiments and compared using either two-tailed independent Student’s t-test or one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Differences were considered to be significant at p < 0.05.