Suberoylanilide hydroxamic acid represses glioma stem-like cells
© The Author(s). 2016
Received: 8 September 2016
Accepted: 3 November 2016
Published: 18 November 2016
Glioma stem-like cells (GSCs) are proposed to be responsible for high resistance in glioblastoma multiforme (GBM) treatment. In order to find new strategies aimed at reducing GSC stemness and improving GBM patient survival, we investigated the effects and mechanism of a histone deacetylases (HDACs) inhibitor, suberoylanilide hydroxamic acid (SAHA), since HDAC activity has been linked to cancer stem-like cell (CSC) abundance and properties.
Human GBM cell lines were plated in serum-free suspension cultures allowed for sphere forming and CSC enrichment. Subsequently, upon SAHA treatment, the stemness markers, cell proliferation, and viability of GSCs as well as cellular apoptosis and senescence were examined in order to clarify whether inhibition of GSCs occurs.
We demonstrated that SAHA attenuated cell proliferation and diminished the expression stemness-related markers (CD133 and Bmi1) in GSCs. Furthermore, at high concentrations (more than 5 μM), SAHA triggered apoptosis of GSCs accompanied by increases in both activation of caspase 8- and caspase 9-mediated pathways. Interestingly, we found that a lower dose of SAHA (1 μM and 2.5 μM) inhibited GSCs via cell cycle arrest and induced premature senescence through p53 up-regulation and p38 activation.
SAHA induces apoptosis and functions as a potent modulator of senescence via the p38-p53 pathway in GSCs. Our results provide a perspective on targeting GSCs via SAHA treatment, and suggest that SAHA could be used as a potent agent to overcome drug resistance in GBM patients.
Glioblastoma multiforme (GBM) has a high recurrence and mortality. The median survival time after a combination of radiotherapy and adjunct temozolomide (TMZ) treatment is only about 14.6 months due to frequent tumor relapse after surgical removal . GBM tumor cells are highly invasive, proliferate rapidly, and easily display resistance to chemotherapy and irradiation. The aggressiveness and high malignancy of GBM are linked to an abundant source of glioma stem-like cells (GSCs) . Cancer stem-like cells (CSCs) are a small population of cancer cells that possess the ability to self-renew and are capable of inducing tumorigenesis . The existence of CSCs has been identified not only in the brain but also in the breast, lung, and colon cancers . GSCs are considered responsible for resistance of GBM to chemotherapy and radiotherapy via their ability to activate the cellular DNA damage response  and to resist radiation-induced apoptosis through an increase in free radical scavenging . In order to develop novel, efficient therapeutics against GBM, it is therefore important not only to target GBM cancer cells but also to enhance cytotoxicity of GSCs.
Histone deacetylases (HDACs) are overexpressed in a wide variety of cancers , and recent studies indicated that HDACs activities are required for maintaining CSC in breast cancer and head and neck tumors [8, 9]. In order to block their oncogenic potential in malignant disease and their function in CSC, HDAC inhibitors are considered to be applied in both experimental and clinical settings. Suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, has been shown to possess cytotoxicity in various cancer cell-types such as T-cell lymphoma , breast cancer , prostate cancer , and glioblastoma cells . A previous study indicated that SAHA elicits anti-cancer effects through the induction of differentiation, apoptosis and cell cycle arrest . However, the pharmacological effects of SAHA on CSCs such as GSCs remain poorly understood.
Cellular senescence is a state of irreversible cell cycle arrest evoked by several oncogenic stimuli. It includes a tumor-suppressive mechanism . Senescence in tumor cells is usually associated with a DNA-damage response and with tumor suppressor pathways such as p53-p21 and p16-pRb . p38 also plays an important regulatory role in inducing cellular senescence, which can be triggered by Ras-Raf activation, oxidative stress, culture stress, or telomere shortening . Furthermore, p38 is shown to be important in mediating oncogene-induced senescence in fibroblasts, indicating a role in tumor suppression . Replicative immortality, which is one of the hallmarks of cancer, is also a potential therapeutic target [17, 18]. Therefore, eliciting cellular senescence or apoptosis could effectively repress immortality of GBM cancer cells and may be important for overcoming poor prognosis in GBM patients [19–21].
In this study, human GBM cells were plated in serum-free suspension cultures allowed for GSC enrichment [3, 22, 23]. We examined the effects of SAHA on cell proliferation, viability, and stem cell-related markers expression in GSCs. In addition, we studied GSCs reduction induced by SAHA through the mechanism of cellular apoptosis and senescence by performing flow cytometry analysis and senescence-associated β-galactosidase (SA-β-gal) assay respectively. By examining the signaling pathway, we further understood the mechanism of action of SAHA. Our results show that SAHA is a potential candidate for treating aggressive and therapy-resistant forms of GBM.
Human glioblastoma U87MG and U373MG cells were obtained from American type culture collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (GIBCO, 12800-017) supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2. For sphere culture, cells were trypsinized using 0.05% trypsin/EDTA and suspended at a concentration of 1.6 × 105 cells in 6-cm dishes pre-coated with poly-(2-hydroxyethyl methacrylate) (poly-HEMA) for 2–14 days. The cells were cultured in serum-free DMEM/F12 (GIBCO, 12500-062) containing 10 ng/ml EGF (Invitrogen, PHG0311), 10 ng/ml FGF (Invitrogen, PHG0263) and N-2 supplement (GIBICO, 17502-048). After spheroid formation, the spheres were pre-incubated with or without 3 μM SB203580 for 1 h and treated with different doses of SAHA for various time intervals as described in figure legends. The morphology of spheroid bodies was observed using light microscopy.
Chemicals and antibodies
SAHA was synthesized by Prof. J. P Liou in Taipei Medical University  and was dissolved in dimethylsulfoxide (DMSO) (Fisher Scientific, 67-68-5). SB203580, a specific p38 inhibitor, was purchased from Selleckchem (S1076) and dissolved in DMSO. The following commercial antibodies were used in Western blotting and Immunofluorescence: CD133 (Proteintech, 18470-1-AP), Bmi1 (GeneTex, GTX114008), Nanog (GeneTex, GTX100863), Oct3/4 (Santa Cruz, SC5279), Vimentin (GeneTex, GTX100619), Sox2 (GeneTex, GTX101507), tubulin (Proteintech, 66031-1-lg), PARP (Cell Signaling, 9542S), caspase-8 (GeneTex, GTX110723), caspase-9 (Cell Signaling, 9502S), caspase-3 (Cell Signaling, 9661S), cyclin B1 (Santa Cruz, SC245), CDK1 (Santa Cruz, SC8395), p21 (Cell Signaling, 2947S), PCNA (Cell Signaling, 2586S), Phospho-p38 (p-p38, T180/Y182) (Cell Signaling, 9211S), p38 (Cell Singling, 8690S), p53 (Cell Signaling, 2524S), Phospho-p53 (Ser15) (Cell Signaling, 9284S), Phospho-p53 (Ser33) (Cell Signaling, 2526S), and Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibodies (Invitrogen, A-11037).
MTT viability/proliferation assay
Cells or dissociated spheroids by trypsin/EDTA were incubated with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (final concentration of 0.5 mg/ml, Bionovas, AM0815-0001) for 1 h at 37 °C. After incubation, the MTT solution was removed and the resultant formazan crystals were dissolved in 200 μl DMSO. The absorbance readings of DMSO extracts were measured at 570 nm using the iMark Microplate Absorbance Reader (Bio-Rad).
Western blotting analysis
GSCs were lysed using modified RIPA buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 15 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40 and 0.1% sodium deoxycholate) containing a protease inhibitor cocktail (Roche, 04693116001). The lysates were centrifuged at 13,200 rpm for 20 min at 4 °C and supernatants were collected for quantification of proteins by Micro BCA Protein Assay Kit (Thermo scientific, 23235). Equal amounts of proteins from each sample (10–20 μg) were separated by 10–12% SDS-PAGE and transferred onto a PVDF membrane (Bio-RAD, 162-0177). The membrane was blocked with TBST buffer containing 5% nonfat milk, and then was incubated overnight with appropriate primary antibodies at 4 °C. Finally, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using an ECL kit (GE Healthcare, RPN2106) and visualized on a Kodak autoradiography film.
Cells were fixed with 4% paraformaldehyde for 20 min, blocked with 1% BSA in PBS for 1 h, and incubated with 1:100-diluted primary antibodies for 4 h at 4 °C. Next, the cells were washed with PBS and incubated with 1:200-diluted Alexa Fluor 594-conjugated second antibodies for 1 h at 4 °C. Finally, the cells were mounted in glycerol containing 4’-6-diamidino-2-phenylindole (DAPI), and examined using a fluorescence microscope (Leica STP6000).
Senescence-associated β-galactosidase (SA-β-gal) assay
Spheroid GSCs were fixed with 1× fixation buffer provided in an SA-β-gal assay kit (Cell signaling, 9860) for 10–15 min. After fixation, the cells were centrifuged at 800 rpm at room temperature for 3 min, following by manufacturer’s guidelines. For each condition, three bright-field micrographs were captured and counted in three randomly chosen fields. All cultures were performed in triplicate and represented by three independent experiments.
Soft agar colony formation assay
To examine anchorage-independent growth, trypsinized spheres (2 × 104 cells/ml) were suspended in 0.4% SeaKem LE Agarose (Lonza, 50004) with serum-free DMEM/F12 containing 10 ng/ml EGF, 10 ng/ml FGF and N-2 supplement. This suspension formed the upper layer, which was poured onto a lower layer formed of 0.8% agarose in a composition otherwise identical to the upper layer. The layers with the cell suspension were poured into 6-well plates. The cells were incubated at 37 °C in a 5% CO2 atmosphere. After 14–30 days, the cells were stained with 0.05% crystal violet, and the colonies were photographed and counted in three randomly chosen fields. All cultures were performed in triplicate and represented by three independent experiments.
Annexin V-PI staining
The spheres were centrifuged at 800 rpm for 3 min and trypsinized with 0.05% trypsin/EDTA in order to resuspend them. An FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, 556547) was employed according to the manufacturer’s guidelines. Flow cytometry was carried out using a BD FACSCalibur Flow Cytometer.
Total RNA was isolated by TRIzol (Thermo Fisher Scientific, 15596026), and 3 μg of total RNA were subjected to SuperScript II Reverse Transcriptase PCR (Thermo Fisher Scientific, 18064014). The expression of p53 mRNA was determined using 2 × SYBR real time master mix (Applied Biosystems) and primers specific to p53 (F: 5'- CTTTGAGGTGCGTGTTTGTG; R: 5'- GTGGTTTCTTCTTTGGCTGG), or the control gene GAPDH (F: 5’- GAAGGTGAAGGTCGGAGTC; R: 5’- GAAGATGGTGATGGGATTC). SYBR green fluorescence was then monitored using an ABI 7000 Sequence Detection System (Applied Biosystems).
The data collected from each experiment, containing both experimental and control groups, were reported as means while error bars represent the standard deviation (S.D.). One-way analysis of variance and an unpaired Student’s t-test were performed to analyze the differences between groups using the GraphPad Prism 5 program. *, P < 0.05; **, P < 0.01 are considered statistically significant. The quantification of protein expression was done using band analysis tools in imageLab software 5.2.1 (Bio-RAD). The band intensities obtained from Western blotting were quantified by subtracting background density and normalizing against the loading control.
SAHA suppresses cell viability/proliferation and stemness-related markers in GSCs
SAHA induces apoptosis in U87MG GSCs
SAHA at a low concentration leads to cell cycle arrest in GSCs
Low concentrations of SAHA induce premature senescence in GSCs via p38 activation
Self-renewal in CSCs fuels tumor growth and provides the maintenance of stemness in a small population of cancer cells [3, 38]. If a therapeutic treatment eliminates the non-self-renewing cells, but not the CSCs. In this case, the tumor would shrink, but it would return form the remained CSCs when the therapy is stopped. Therefore, preventing drugs resistance and self-renewal in GSCs are important to avoid GBM recurrence. As shown in Fig. 2, we found that GSCs were inhibited upon SAHA treatment, suggesting that SAHA may overcome therapeutic resistance in GBM by targeting GSCs. Furthermore, the marker Bmi1 is an important factor for the self-renewal and stemness maintenance of CSCs . This suggests that targeting Bmi1 may be an efficient way to prevent cancer recurrence. As shown in Fig. 2, SAHA significantly suppressed Bmi1 expression in a dose-dependent manner, implying that SAHA inhibits GSCs by blocking the self-renewal mechanism. In addition, we have examined the viability between U87MG adherent cells and U87MG spheroids by performing MTT assay, and verified SAHA at higher doses, primarily induces apoptosis, leading to inhibition of cellular proliferation in both cells (Fig. 2a and b). Interestingly, at low-dose treatment of SAHA (1 μM), only spheroids were inhibited well up to 6 days after treatment comparing with U87MG adherent cells. The result implies that GSCs are sensitive to SAHA-induced senescence.
Previous study indicated that p53 plays an essential role in SAHA-induced apoptosis in MCF-7 cells , whereas other studies suggested that SAHA shows preferential cytotoxicity in several p53-mutant cancer cells through inhibition of the HDAC6-Hsp90 chaperone axis , indicating the complexity of mechanism by which SAHA medicates apoptosis in wild type and p53-deficient tumors. In cancer stem cell-like spheroids, we found that both levels of p53 protein and its phosphorylation at Ser15, but not at Ser33, were increased within 2 days after treatment with high doses (10 μM and 25 μM) of SAHA (Fig. 5h). Especially, these doses of SAHA triggered significant apoptosis in U87MG (p53wt) GBM cells (Fig. 3b). When we further used another malignant GBM cell line, U373MG that expresses mutant p53 protein (R273H mutation, p53R273H), however, a high-dose SAHA (10 μM) inhibited cell viability in U373MG spheroids as well (Additional file 1: Figure S1). These results indicated that SAHA inducing cell death in cancer cells and even in cancer stem cell-like spheroids might be through both p53-dependent and p53-independent apoptosis pathways. A recent study further investigated the mechanism of SAHA-induced apoptosis in couple leukemic cell lines, HL-60 (p53null) and CML-T1 (p53wt) . While leukemic cells were treated with combination of SAHA and decitabine (DAC), a methyltransferase inhibitor, p53-dependent apoptosis is triggered by the upregulation of Puma and p21 in CML-T1 cells, and p53-independent apoptosis is triggered by inhibition of survivin and upregulation of p21 in HL-60 cells. Although this study probably explained the different mechanisms of SAHA-induced cell death between p53null and p53wt cancer cells, in GSCs the roles of p53 after SAHA treatment remains unclear and needs further studies.
Since many post-translational modifications reportedly modulate protein stabilization and transcriptional activation of p53 , the rigorous control of these modifications on p53 may determine its functional role under several stresses. Various p53 residues can be phosphorylated by different kinases. For example, phosphorylation at Ser 6, 9, and 15, is carried out by ATM, occurs in response to genotoxic stresses, especially Ser15 of p53 is rapidly phosphorylated after stress in order to represent a priming event for the subsequent series of modifications , such as Ser20 phosphorylation by Chk2 and C-terminal acetylation by p300, CBP and P/CAF. Then these secondary modifications prevent the degradation of p53 and increase its apoptotic induction [43, 45]. Furthermore, stressful situations may also elevate p53 phosphorylation at Ser33, by p38 kinase, and at Thr81, by JNKs kinase, all of which are present in Ser/Thr–Pro motifs. These phosphorylated motifs lead to p53 interacting with Pin1 which in turn induces proline isomerization in p53 . Such conformational changes have intense effects on p53 transcriptional activity and protein stability and shows an increase in cell cycle checkpoint activation following genotoxic stress. Therefore, it is evident that only a number of stress-responsive kinases alter p53 at exact sites which recruits Pin1 interaction in order to complete an additional step in the process of activation.
When we treated GBM stem cell-like spheroids with low doses of SAHA (1 μM and 2.5 μM), we found that both p53 protein and its phosphorylation at Ser33 were slight increased on 2 days after SAHA treatment, but they were risen significantly after 1 week of treatment (Fig. 5g and h). However, pharmacological inhibition of p38 kinase attenuated p53 accumulation and its phosphorylation at Ser33, which resulted in a reduction in the senescent phenotype (Fig. 5d to g). In addition, we observed that low doses of SAHA-induced phosphorylated p53 at Ser33 didn’t seem to correlate with apoptosis occurrence by the detection of caspase-3 activity (Fig. 5g and h). Therefore, we summarized the results revealing that low-dose and long-term treatment with SAHA in GBM spheroids induces p53 phosphorylation at Ser33 causing an increase in cell cycle checkpoint activation (Fig. 6), but SAHA in high-dose and short-term treatment induces p53 phosphorylation at Ser15 leading to p53-dependent apoptosis in U87MG spheroids, and even induces p53-independent apoptosis in U373MG spheroids (Fig. 6).
To further understand the regulated mechanism of p53 after SAHA treatment, Real-time PCR was performed and the result showed a slight increase in p53 mRNA levels after low concentrations treatment of SAHA, but no change at a high-dose (10 μM) treatment (Additional file 1: Figure S3). Combining with the reason that phosphorylation of p53 at multiple sites, including Ser15, Ser20, and Ser33, can protect its protein stability against MDM2-mediated ubiquitination and degradation , we think that low-doses SAHA might alter the transcriptional activity and protein stability to trigger p53 up-regulation, but high-doses SAHA rise p53 levels primarily by preventing the protein degradation.
Cellular senescence is a tumor-suppressive mechanism leading to permanent cell cycle arrest. Although SAHA-induced polyploidy, resulting in loss of proliferation and commitment to senescence, has been demonstrated in human colon cancers and breast cancer cells , the effects and molecular mechanisms of SAHA in CSCs remained unclear. In our study, we clarified that a reduced dose of SAHA not only increases polyploidy but also decreases S and G2/M cells in GSCs, indicating that SAHA induces GSCs senescence by blocking cell cycle progression as well as via polyploid cell accumulation. Previous study indicated that Cyclin A1 is a p53-induced gene that mediates cycle arrest and polyploidy . Additionally, we used a p38 inhibitor to demonstrate that p38-regulated p53 phosphorylation at Ser33, affecting p53 transcriptional activation and protein accumulation, is essential for SAHA-induced GSC senescence. Crucially, SAHA has been determined capable of crossing the blood-brain barrier at micromolar concentrations [49, 50]. Taken together, the evidence suggests that at lower doses, SAHA may be used as a drug targeting CSCs through the induction of senescence, helping to overcome drug resistance in GBM.
We demonstrated that SAHA suppressed cell proliferation and diminished stemness properties in GSCs. Administration of high concentrations of SAHA triggered apoptosis of GSCs accompanied by increases in both activation of caspase 8- and caspase 9-mediated pathways. Interestingly, we found that SAHA at a low dose led to cell cycle arrest in GSCs via upregulation of p21, and induced premature senescence through activating p38/p53 pathway. Our results provide a perspective on targeting GSCs via SAHA treatment, and suggest that SAHA could be used as a potent agent to overcome drug resistance in GBM patients.
This work was supported by grants from the Ministry of Science and Technology (MOST 103-2320-B-038-046-MY3, MOST 105-2320-B-038-063, MOST 103-2314-B-400-011-MY3, MOST 105-2911-I-038-503, and MOST 104-2923-B-038-002-MY3), the Ministry of Health and Welfare (MOHW 105-TDU-B-212-134001), and the Ministry of Education/Taipei Medical University (TMUTOP103006‐9).
Availability of data and materials
CCH, TIH, and JJL carried out experiments, collected and analyzed data, prepared the manuscript. WCC participated in designing the experiments and reviewing the manuscript. SHY, JYW, JPL, and CYK contributed for data analysis and reviewed the manuscript. KYC and JYC conceived experiments and contributed for analyzing data, preparing and reviewing the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Our study don’t include any data about people or human tissue samples.
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