Napabucasin, a novel STAT3 inhibitor suppresses proliferation, invasion and stemness of glioblastoma cells
Glioblastoma (GBM) cells with stem cell-like properties are called glioma stem cells (GSCs). GSCs display highly treatment resistance and are responsible for tumor recurrence. Napabucasin (BBI608), a novel small molecule inhibitor of STAT3, has been identified to eliminate stemness-like tumor cells in some cancers. However, the influence of Napabucasin on GBM cells, especially on GSCs, is currently unclear. In this study, we explored the influence and underlying mechanisms of Napabucasin on GBM cells.
STAT3 expression and its correlation with the glioma grade and patient survival were analyzed using CGGA and TCGA glioma databases. The influence of Napabucasin on proliferation, stemness, the cell cycle, apoptosis, and invasion of human GBM cell lines U87MG and LN229 was tested by CCK8, EdU incorporation, colony formation, Transwell invasion, and three-dimensional spheroid assays as well as flow cytometry, qPCR, and western blot analysis. The ability of Napabucasin to inhibit cell proliferation of U87MG tumor xenografts in mice was assessed using a live animal bioluminescence imaging system and immunohistochemistry.
Napabucasin suppressed the proliferation, colony formation, and invasion of U87MG and LN229 cells. Furthermore, Napabucasin induced cell cycle arrest and apoptosis. More importantly, Napabucasin treatment obviously inhibited expression of stemness-associated genes including STAT3 and suppressed the spheroid formation of glioma cells in vitro. Napabucasin also disrupted the NF-κB signaling pathway via downregulation of RelA (p65). Finally, glioma growth was effectively impaired by Napabucasin in nude mice bearing intracranial glioma xenografts.
Napabucasin treatment may be a novel approach for the treatment of GBM, particularly GSCs.
KeywordsGlioma STAT3 Inhibitor Cancer stem cell
Glioma stem cell
Half maximal inhibitory concentration
Signal transducer and activator of transcription 3
Glioblastoma (GBM) is the most common and heterogeneous primary brain tumors in adults, accounting for more than 50% of glioma cases. It is also one of the most lethal cancers and challenging to treat . Comprehensive standard therapies for GBM are maximal surgical resection, radiation, and chemotherapy using the alkylating drug temozolomide. Although many aggressive therapeutic methods are employed, the prognosis for GBM patients remains poor with median overall survival (OS) ranging from 14.6 to 16.8 months [2, 3]. Despite the further characterization of the distinct molecular alterations in GBMs by large-scale gene-expression studies, multiple clinical trials of novel therapies for GBM patients have failed to improve OS . Glioma stem cells (GSCs) are defined by their ability for self-renewal, multilineage differentiation, and tumorigenicity meditated by various signaling pathways responsible for treatment resistance in GBMs . Chemotherapy resistance is an intrinsic property of GSCs, which is acquired via multiple independent mechanisms including an increase of drug efflux pumps, an enhanced DNA repair capacity, and protection against reactive oxygen species . Because GSCs contribute to glioma initiation, propagation, and recurrence, they are a crucial target of anti-GBM therapies.
Various molecular signaling pathways have been identified as prognostic markers or therapeutic targets for GBM . Dysregulation of signal transducer and activator of transcription 3 (STAT3), a classic oncogenic transcription factor regulating the expression of a wide range of genes, has been reported in 50–90% of all human cancers including GBM . Abundant evidence has highlights the essential roles of STAT3 in GSCs [9, 10]. Furthermore, STAT3-targeting agents to generate potent anti-glioma effects in the clinic remain to be further explored. Napabucasin (BBI608) is a newly developed small molecule inhibitor of STAT3, which has been shown to impair self-renewal and induce apoptosis in cancer stem cells of colorectal, pancreatic, non-small cell lung, gastric, and prostate cancers [11, 12, 13, 14, 15]. Importantly, Napabucasin is an orally administered agent. Clinical trials have been performed, many of which were in combination with numerous chemotherapeutic agents [16, 17, 18].
In this study, we found that Napabucasin effectively suppressed the proliferation, invasion, and sphere formation ability of GBM cells. It also arrested the cell cycle and induced apoptosis of GBM cells in vitro. Napabucasin decreased the expression of STAT3 and stemness-associated genes. Furthermore, Napabucasin displayed potent activity against tumor growth in an orthotopic nude mouse model of GBM. Taken together, Napabucasin treatment may be a novel approach to suppress GBM progression and improve its prognosis.
This study was performed in accordance with the ethical standards according to the Declaration of Helsinki and national and international guidelines and was approved by The Research Ethics Committee of Nanjing Medical University.
Cell culture and STAT3 inhibitor
Human GBM cell lines U87MG and LN229 used in this research were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, UT, USA) supplemented with 100 units of penicillin/mL, 100 ng of streptomycin/mL, and 10% fetal bovine serum (Gibco, MD, USA). These cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Cell lines were routinely tested for absence Mycoplasma. Napabucasin was purchased from MedChem Express (New Jersey, USA).
Cell counting kit-8 assay
U87MG and LN229 were plated in 96-well plates (2000 cells/well) in triplicate and were incubated at 37 °C overnight. Subsequently, the cells were treated with Napabucasin (1, 5, and 10 μM) or vehicle control (0.1% DMSO) for 24, 48, and 72 h. Finally, the cell growth was measured by CCK-8 Cell Counting Kit (Dojindo, Japan) following the manufacturer’s protocols. Briefly, after one-hour incubation with CCK-8 at 37 °C, OD value (450 nm) was detected for calculating cell viability.
5-ethynyl-29-deoxyuridine (EdU) proliferation assay
To test the growth activity of U87MG and LN229 cells, Click-iT EdU Alexa Fluor 594 Imaging Kit (Thermo Fisher Scientific, Massachusetts, USA) was used according to the manufacturer’s protocol. After treated with 5 μM GSK343 or 0.1% DMSO for 48 h, the proportion of cells incorporated EdU was determined with fluorescence microscopy (Nikon, Tokyo, Japan).
Colony formation assay
For the colony formation assay, cells were seeded in six-well plates (300 cells/well) and cultured for approximately 2 weeks until colony formation was observed. Colonies were fixed with methanol and stained with 1% crystal violet (Sigma, USA). The number of colonies was counted to determine colony-forming efficiency.
Wound healing assay
U87MG and LN229 cells were seeded in 6-well plates and were cultured until reached 100% confluence. Next, a ten-microliter sterile pipette tip was used to create scratches across the cell monolayer and the photographs of scratched areas were taken. Then, cells were treated with 1 μM Napabucasin or vehicle control. One day later, a total of nine scratched areas were selected randomly in each well and were photographed under an inverted microscope. The cells protruding from the border of the scratches were counted.
Transwell invasion assay
24-well plates using Transwell inserts (Corning, New York, USA) which were pre-coated with Matrigel (BD Biosciences, New Jersey, USA) were used to measure cell invasion. The upper chambers were added with 50,000 U87MG and LN229 cells which were treated with 1 μM Napabucasin or 0.1% DMSO and were dissolved in 200 μL serum-free media. In parallel, 900 μL DMEM media with 10% FBS was added to the lower chamber of each well. After incubation for 24 h at 37 °C, cells from the upper surface of the membrane were removed with a cotton swab and the penetrated cells were fixed with 4% methanol for 5 min and then stained with 0.1% crystal violet for 30 min. In each well, six fields of cells were captured and counted under an inverted microscope.
Three-dimensional spheroid assays
U87MG and LN229 cells were seeded at a 2000 cells/ml density in 96-well ultra-low adherence plates (Corning, New York, USA). Over the course of 96 h these cells were induced to aggregate into a multicellular spheroid and then Matrigel mixed with 1 μM Napabucasin or 0.1% DMSO was added into wells. After 48 h, the motion of cells was confirmed as fully formed under light microscopy.
Flow cytometry analysis
U87MG and LN229 cells were harvested and washed with phosphate-buffered saline (PBS). Then, 75% ethanol was used to fix cells at − 20 °C overnight. Next, DNA in cells was stained by Hank’s balanced salt solution including 50 mg/mL propidium iodide and 50 mg/mL RNaseA for 1 h at room temperature. Finally, the cell cycle of these cells was analyzed by Gallios flow cytometer (Beckman Countler).
RNA extraction and quantitative real-time PCR
RNA was extracted from cells by TRIzol reagent (Invitrogen, Darmstadt, Germany). TaqMan real-time reverse transcription PCR was carried out for synthesizing cDNA by ABI StepOnePlus system (Applied Biosystems, Massachusetts, USA). Then, quantitative real-time PCR analysis was performed using ABI PRISM 7500 FAST Real-TIME PCR System. The relative expression was determined through the CT method and the results were normalized to GAPDH expression.
GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′;
STAT3, forward 5′-CAGCAGCTTGACACACGGTA-3′ and reverse 5′-AAACACCAAAGTGGCATGTGA-3′;
β-catenin (CTNNB1), forward 5′-AAAGCGGCTGTTAGTCACTGG-3′ and reverse 5′-CGAGTCATTGCATACTGTCCAT-3′;
SOX2, forward 5′-GCCGAGTGGAAACTTTTGTCG-3′ and reverse 5′-GGCAGCGTGTACTTATCCTTCT-3′;
OCT4 (POU5F1), forward 5′-GTGTTCAGCCAAAAGACCATCT-3′ and reverse 5′-GGCCTGCATGAGGGTTTCT-3′;
NESTIN, forward 5′-CTGCTACCCTTGAGACACCTG-3′ and reverse 5′-GGGCTCTGATCTCTGCATCTAC-3′;
Western blot analysis
RIPA lysis buffer (KeyGEN, Jiangsu, China) was used to extract proteins from cells. Equal amounts of protein were separated by SDS-PAGE, followed by electro-transfer onto poly-vinylidene difluoride membranes (Thermo Fisher Scientific, Massachusetts, USA). Then, membranes were blocked for 2 h with 5% nonfat milk and then incubated at room temperature with the primary antibody. After incubating with secondary antibody for 2 h, membranes were developed using an enhanced chemiluminescence detection system (GE Healthcare, Chicago, USA). All the antibodies were obtained from Cell Signaling Technology (Massachusetts, USA).
Cells fixed by 4% formalin were incubated with rabbit monoclonal anti-cleaved caspase-3 overnight at 4 °C and then with Alexa 488-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific, Massachusetts, USA) 2 h at room temperature. After treated with DAPI (Beyotime Biotechnology, Jiangsu, China) for 10 min, cells were examined with Zeiss Axiophot photomicroscope (Carl Zeiss AG, Jena, Germany).
In vivo experiments, IHC, and H&E staining
Animal experiments were approved by the Animal Management Rule of the Chinese Ministry of Health (documentation 55, 2001) and were in accordance with the approved guidelines and the experimental protocol of Nanjing Medical University. 6-week-old female nude mice purchased from Cancer Institute of the Chinese Academy of Medical Science were implanted with 1 × 106 luciferase-expressing U87MG cells per mouse (Napabucasin-treated group, N = 6; DMSO-treated group, N = 6). After 7 days, nude mice received either vehicle (10 μL DMSO in 200 μL PBS) or Napabucasin (40 mg/kg in 200 μL PBS) intraperitoneally every other day. Tumor growth was assessed weekly by live animal bioluminescence imaging system.
To analyze differences in each two-group comparison, t-test was employed while one-way ANOVA was performed to determine the difference among at least three groups. Kaplan–Meier analysis was used to assess the survival rate of mice. P < 0.05 was considered statistically significant.
Napabucasin inhibits glioma cell proliferation and represses the expression of STAT3
Napabucasin arrests the cell cycle in GBM cells and induces apoptosis
Napabucasin represses the migration and invasion of GBM cells
Napabucasin impairs the stemness of GBM cells
Napabucasin downregulates the expression and phosphorylation of RelA
Napabucasin suppressed tumor growth in an orthotopic glioma model
Although current standard treatments for glioblastoma (GBM, WHO IV) are able to eliminate the majority of tumor cells, recurrence is still inevitable . Many studies have indicated that harboring glioma stem cells (GSCs), a small subpopulation of GBM cells with unlimited self-renewal and the capacity to regenerate tumorigenic progenies, may be a major cause of disease relapse . Therefore, targeting GSCs is a promising and efficacious strategy to eradicate GBM. Signal transducer and activator of transcription 3 (STAT3) is an essential transcription factor that controls many physiological and pathological processes including the cell cycle, cell survival, angiogenesis, and immune responses. Considering the well-documented role of overactive STAT3 signaling in glioma, the therapeutic potential of targeting this pathway should be emphasized. Multiple attempts to develop inhibitors against STAT3 pathway have been reported, and a variety of STAT3 inhibitors, including chemicals, STAT3-binding peptides, and siRNA reagents, have been developed with various degrees of success [21, 22].
Napabucasin (BBI608), a novel STAT3-specific inhibitor, was identified by Li et al. . They revealed that Napabucasin efficiently suppressed metastasis and relapse of a variety of cancers by inhibition of STAT3-driven gene transcription. Importantly, Napabucasin treatment impairs spheroid formation of liver cancer stem cells and downregulates the expression of stemness genes such as SOX2, BMI-1, Nanog, and c-Myc. Considering the promising preclinical data of Napabucasin as both a monotherapy and in combination with conventional chemotherapeutic methods, several clinical trials have been performed . Furthermore, a phase III trial of Napabucasin for refractory colorectal cancer highlighted STAT3 as an essential target for the treatment of patients with elevated pSTAT3 expression . However, the effect of Napabucasin on GBM and GSCs remains unclear.
To our knowledge, this is the first study to evaluate the influence of Napabucasin in the context of GBM. We confirmed that STAT3 is a promoting factor for malignant progression and poor prognosis in glioma patients. We found that treatment of GBM cell lines (U87MG and LN229) with Napabucasin significantly blocked cell proliferation, migration, invasion, and sphere formation. Additionally, Napabucasin arrested the cell cycle and induced apoptosis of GBM cells. Following treatment with Napabucasin, the protein and mRNA expression of STAT3 and stemness-associated genes were obviously decreased. Napabucasin treatment also significantly reduced the expression and phosphorylation of RelA, a key transcription factor of the NF-κB signaling pathway. Moreover, Napabucasin repressed tumor growth in an orthotopic nude mouse model of GBM. One of the advantages of Napabucasin is that it remarkably decreased the protein level of STAT3 and did not simply inhibit the phosphorylation of STAT3. In a study by Zuo et al., the underlying mechanism of the downregulated STAT3 protein level was mediated by protein synthesis inhibition induced by Napabucasin . In our study, we reveal that Napabucasin treatment reduced STAT3 expression at the transcriptional level. Overall, our results support the potential use of Napabucasin as an efficacious anti-glioma therapeutic agent.
Our results indicate that Napabucasin suppresses the expression of STAT3, and thus impairs the proliferation, migration, and invasion of glioblastoma cells. Napabucasin administration effectively decreases the stemness of glioma cells. Furthermore, we found that Napabucasin significantly impairs the expression and phosphorylation of RelA, which is a novel pharmacological mechanism of Napabucasin. Importantly, treatment with Napabucasin impedes the growth of intracranial U87MG-derived tumors in nude mice. Therefore, Napabucasin may be a promising and potent agent to suppress GBM progression.
This work was supported in part by grants from the National Natural Science Foundation of China (81501138).
DFH performed the experiments, analyzed the data and drafted the manuscript. ND, BW and FS performed the experiments. DHJ planned and supervised the study as well as reviewed the manuscript. DFH and TFY revised the manuscript. All authors read and approved the final manuscript.
All animal experiments were approved by the Ethical Committee of Xuzhou central hospital.
Consent for publication
The authors declare that they have no competing interests.
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