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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 39-47

Panobinostat and its combination with 3-deazaneplanocin-A induce apoptosis and inhibit In vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines

1 Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain
2 Molecular Pathology Research Unit, Virgen de la Salud Hospital, Toledo, Spain
3 IdiPaz Research Unit, La Paz University Hospital, Madrid, Spain
4 Department of Pathology, University of Navarra Clinic, Pamplona, Spain

Date of Submission10-Apr-2018
Date of Acceptance17-Apr-2018
Date of Web Publication27-Apr-2018

Correspondence Address:
Javier S Castresana
Department of Biochemistry and Genetics, University of Navarra School of Sciences, Irunlarrea 1, 31008 Pamplona
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_12_18

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Aim: Glioblastoma is the most malignant primary brain tumor. The treatment consists of surgery, with or without radiotherapy, and temozolomide, with a life expectancy of 12–15 months after diagnosis. Glioblastoma is resistant to conventional antitumor therapies. In this work, we present a preliminary in vitro study of two epigenetic drugs against GOS-3 glioblastoma cells.
Methods: We used (1) panobinostat, a histone deacetylase inhibitor, and (2) 3-deazaneplanocin-A (DZ-Nep), an inhibitor of enhancer of zeste homolog 2 (EZH2) (enzyme of the polycomb repressor complex 2, polycomb group of proteins that trimethylate lysine 27 of histone 3-H3K27 me3-), as treatments that might modulate the PI3K pathway, affected in GOS-3 cells due to PTEN haploinsufficiency. The glioblastoma cell line GOS-3 was exposed to DZ-Nep and panobinostat treatments, separately and in combination, over a period of 2 days, after which cell migration, clonogenicity, and molecular expression characterization assays were performed.
Results: Panobinostat alone or the combination of panobinostat plus DZ-Nep inhibited clonogenicity, metastasis, angiogenesis, epithelial–mesenchymal transition, and entry in the S phase of the cell cycle and induced apoptosis in GOS-3 glioblastoma cells. On the contrary, DZ-Nep inhibited cell migration (single treatment) and O(6)-methylguanine-DNA methyltransferase expression (DZ-Nep or double treatment).
Conclusion: Panobinostat alone or the combination of panobinostat and DZ-Nep induce apoptosis and inhibit in vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines.

Keywords: 3-Deazaneplanocin-A, enhancer of zeste homolog 2, epigenetics, histone deacetylases, panobinostat

How to cite this article:
de la Rosa J, Urdiciain A, Aznar-Morales JJ, Meléndez B, Rey JA, Idoate MA, Castresana JS. Panobinostat and its combination with 3-deazaneplanocin-A induce apoptosis and inhibit In vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines. Cancer Transl Med 2018;4:39-47

How to cite this URL:
de la Rosa J, Urdiciain A, Aznar-Morales JJ, Meléndez B, Rey JA, Idoate MA, Castresana JS. Panobinostat and its combination with 3-deazaneplanocin-A induce apoptosis and inhibit In vitro tumorigenesis and metastasis in GOS-3 glioblastoma cell lines. Cancer Transl Med [serial online] 2018 [cited 2019 Jan 16];4:39-47. Available from: http://www.cancertm.com/text.asp?2018/4/2/39/231376

*Javier de la Rosa and Alejandro Urdiciain contributed equally to this work and were co-first authors

  Introduction Top

The survival of patients suffering from glioblastoma, even if treated with chemotherapy, is around 15 months.[1],[2] This result has not changed much in the last three decades, despite the existence of new treatments. Standard treatments include surgical resection, radiotherapy, and chemotherapy (temozolomide). Although metastases outside the central nervous system are uncommon, glioblastomas infiltrate brain tissue [3] far beyond the radiographic borders of the tumor, which prevents healing with surgical resection alone. Radiation and chemotherapy are valuable adjuvant therapies; however, specific cell populations are chemo- and radio-resistant, resulting in the recurrence of the tumor.[4]

It is known that a great accumulation of molecular changes appears in glioblastoma:[5] Alterations at epidermal growth factor receptor (primary glioblastomas), p53 or isocitrate dehydrogenase 1/2 (secondary glioblastomas), p14, MDM2, cyclin-dependent kinase inhibitor 2A (CDKN2A) (p16), retinoblastoma, and PI3K-(PTEN)-AKT-mTOR pathway are common.[6] PTEN is a lipid phosphatase that antagonizes the action of PI3K,[7] dephosphorylating PIP3 to generate PIP2 (thus blocking the PI3K signaling cascade). PTEN negatively regulates PI3K activity in the AKT signaling pathway, which is activated by growth factors. In the absence of PTEN, proliferation, cell motility, migration, and invasion increase, due to AKT and mTOR activation, with the subsequent increase in cell division.

Brain tumors are developed not only by gene mutation but also by epigenetic changes, mainly DNA methylation, histone acetylation, histone methylation, and microRNA deregulation.[8],[9],[10],[11],[12] Unlike genetic alterations, epigenetic changes are reversible, and this is the reason why they are attractive targets for cancer therapy.

Histone acetylation is positively regulated by histone acetyltransferases, which induce a relaxed status of chromatin and expression of adjacent genes; and by histone deacetylases (HDACs), which condense the chromatin and stop gene expression. One strategy against cancer nowadays is the use of inhibitors of HDAC, such as panobinostat.[13],[14],[15],[16],[17],[18] Inhibition of deacetylation of histones would procure a relaxed chromatin status of adjacent genes, breaking some pathways which play an important role in cancer development.[19]

Histone methylation is executed by polycomb proteins of the polycomb repressor complex 2 (PRC2). PRC2 consists of three essential core subunits: enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 homolog (SUZ12). EZH2 is the catalytic subunit that carries out the trimethylation of lysine 27 of histone H3 (H3K27 me3), turning off several target genes.[20],[21],[22],[23] EZH2 is overexpressed in different types of cancer, such as prostate,[24] breast,[25] and brain,[25],[26] conferring poor prognosis, chemoresistance, and greater tumor aggressiveness. One possibility to treat tumors that overexpress EZH2 is by its inhibitor, 3-deazaneplanocin-A (DZ-Nep), which is able to interrupt PCR2 activity by inhibiting the H3K27 trimethylation performed by EZH2.[26],[27]

GOS-3, the cell line we used for our experiments, was for decades considered as an astrocytoma/oligodendroglioma cell line, Grades II/III. We and others considered it as such in the previous publications.[28],[29] However, it was recently redefined as a glioblastoma cell line,[30] derived from the U343MG glioblastoma cell line, as indicated by the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures) after GOS-3 fingerprinting analysis (https://www.dsmz.de/catalogues/details/culture/ACC-408.html).

Of special interest when using the GOS-3 glioblastoma cell line is the fact that this cell line presents hemimethylation of PTEN,[31] which might result in PTEN haploinsufficiency as declared by Ding et al.[32] in prostate cancer. According to this fact, and assuming that GOS-3 does not present p53 mutations, we might infer that, similar to prostate cancer, glioblastoma might present a histone acetylation defect that would provoke a decay in H3K27Ac and an increase in H3K27 me3 caused by EZH2.[32]

We proposed anin vitro study of epigenetic modulation at the level of histone methylases and HDAC. GOS-3 glioblastoma cell line was treated with an HDAC inhibitor (panobinostat), with an inhibitor (DZ-Nep) of the trimethylation of lysine 27 of histone H3 (EZH2), and with the combination of both. Treated cells were compared with nontreated cells, to see the effects of panobinostat and DZ-Nep on cell migration and clonogenicity and on gene expression of different cancer-related genes: BAX and NOXA (proapoptotic genes), SUZ12 and EZH2 (components of the PRC2), PHF1 (PRC2 recruiter), CDH1 (E-cadherin gene), tissue inhibitor of metalloproteinase 3 (TIMP3) (metastasis inhibitor), vasohibin 1 (VASH1) (angiogenesis inhibitor), O(6)-methylguanine-DNA methyltransferase (MGMT) (DNA methyltransferase crucial for genome stability), and CDKN2A (p16, cell cycle inhibitor).

  Methods Top

Cell line

GOS-3 glioblastoma cell line was used for the study.[30] It was obtained from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany. We used RPMI L-GlutaMAX medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) with supplements such as 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.1% amphotericin B, and 4% nonessential amino acids for cell culture. The cell line was maintained in the incubator at 37°C, in an atmosphere with 5% CO2. The subcultures were carried out after reaching a confluence of 80%–90% using trypsinization.

Pharmacologic treatments

Concentrations of the drugs were: DZ-Nep, 5 μM;[27] and panobinostat, 20 nM.[33] Drugs were added as a simple treatment or in a double combination for 3 days. Medium with the treatment was changed every day and contained the same quantity of dimethyl sulfoxide, always with a concentration below 0.1%.

Colony formation assay

This test was performed to evaluate the ability of GOS-3 cells to create cell colonies in monolayer cultures after treatments versus control. This experiment requires less time than the formation of colonies in soft agar since GOS-3 cells grow attached to the tissue culture flasks. One thousand cells were cultured in six-well plates under the four conditions studied (control, panobinostat, DZ-Nep, and the two compounds combined), with 2 mL of medium for 10 days. After this time, the medium was discarded and the plates were washed with 2 mL Dulbecco's phosphate-buffered saline (DPBS). The cells of each plate were fixed with 2 mL of 4% paraformaldehyde for 30 min. They were again washed with 2 mL DPBS and stained with 1% crystal violet (Sigma Aldrich Corporation, St Louis, MO, USA) for 15 min. Finally, the plates were washed and left to dry for the subsequent colony count of each condition.

Soft agar clonogenic assay

For the cells to be grown in agar plates, 13 mL of a 1% agarose gel was mixed with 13 mL of 2X Dulbecco's modified Eagle medium (DMEM) (0.5% final gel concentration), of which 2 mL was added to each well. It was allowed to solidify for 1 h. Subsequently, the second layer was added. For this, 6.5 mL of a 0.4% agarose gel was used together with 6.5 mL of 2X DMEM containing 35,000 cells (0.2% final gel concentration). Two milliliters of the mixture was added per well. Then, 2 mL of culture medium with 10% FBS was added as well and was renewed every 3–4 days. It was left to incubate at 37°C for about 21 days. After that, the colonies were stained with 250 μL of 1% violet crystal (Sigma Aldrich Corporation, St Louis, MO, USA) for 5 min. Samples were washed with H2O to improve visualization of the colonies. To count the colonies, a photograph was taken from each well and it was counted with the colony-forming unit (CFU) free software OpenCFU[34] under the same conditions. The plates were stored at 4°C.

Wound-healing assay

GOS-3 cell monolayers of the four experimental conditions (untreated, treated with 5 μM DZ-Nep, treated with 20 nM panobinostat, and treated with both compounds) were trypsinized with trypsin/ethylenediaminetetraacetic acid (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) and approximately 700,000 cells were cultured per well in two six-well plates with 3 mL of medium each. Plates were left to incubate at 37°C in normoxia with 5% CO2 for about 24 h until reaching high confluence (90%). A scratch was done with a pipette tip in each well. The filling of the wound was observed with the microscope at different times. To analyze cell migration, photographs were taken at 0, 6, 12, 24, 30, 48, and 72 h. Cells were maintained in the incubator at 37°C, in normoxia with 5% CO2. Cell migration was evaluated in both the untreated cells (control) and the cells treated with 20 nM of panobinostat, 5 μM of DZ-Nep, and both. The results were studied using Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA), with which the distance covered by the migrating cells was measured by transforming the images into numerical values. Experiments were repeated three times independently with three replicates per experiment.

Gene expression detection by real-time quantitative polymerase chain reaction

RNA was extracted using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) following the protocol instructions. The concentration and purity of the RNA were determined by NanoDrop spectrometer. Typically, 1 μg total RNA was used to generate cDNA using SuperScript reverse transcriptase enzyme (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) with random primers.

As previously mentioned,[35],[36] real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the SYBR Green PCR MasterMix (Bio-Rad, Hercules, CA, USA), as per the instruction of the Real-Time PCR Multicolor IQ5 System (BioRad, Hercules, CA, USA). Each assay was performed in triplicate. The primer sequences are described in [Table 1].
Table 1: Oligonucleotide sequences and annealing temperatures for reverse transcription quantitative polymerase chain reaction

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Western blot analysis

Western blot analysis was used to measure the expression of H3K27 me3 (methylated histone 3 by the action of the EZH2 histone methylase) compared to protein expression of the housekeeping gene beta-actin. Total proteins were extracted by the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Protein concentration of the samples was determined with the BCA Protein Quantification Kit (Thermo Fisher Scientific, Waltham, MA, USA). Thirty micrograms of total proteins of each treatment was loaded on 12% sodium dodecyl sulfate-polyacrylamide gels and run for 1 h at 120 V. Once proteins were electrophoresed, they were transferred to a nitrocellulose membrane. Membranes were blocked with TBS containing 0.1% Tween 20 and 5% milk powder for 1 h and incubated with the primary antibody, diluted 1:1000 for H3K27 me3 (Merck Millipore, Billerica, MA, USA), and diluted 1:20000 for beta-actin (Sigma, Saint Louis, MO, USA). After overnight incubation of the membrane with the primary antibody, three washes were performed with TBS-Tween 0.1%, and membranes were incubated with the secondary antibody, diluted 1:5000 for the H3K27 me3 experiment, and diluted 1:40000 for beta-actin visualization. Finally, a solution was used to develop the membranes by chemiluminescence with films.

Statistical analysis

The statistical analysis was performed using GraphPad Software (GraphPad Software Inc., San Diego, CA, USA). Results have been obtained for the four experimental groups (control, DZ-Nep, panobinostat, and combined treatment) using one-way ANOVA and the Tukey test. Data are reflected as means ± standard deviation. Results are considered to be significant with respect to the control when P < 0.05 (*), very significant when P < 0.01 (**), and highly significant when P < 0.001 (***). Results are considered to be significant with respect to the joint treatment when P< 0.05 (•), very significant when P< 0.01 (••), and highly significant when P< 0.001 (•••).

  Results Top

Double treatments reduce clonogenicity of GOS-3 glioblastoma cells

Panobinostat and the combined treatment, but not DZ-Nep, clearly reduced the formation of two-dimensional (2D) colonies in GOS-3 [Figure 1]a. To study GOS-3in vitro tumorigenicity, the ability of these cells to form colonies in soft agar, without adhesion to a surface, was also studied [Figure 1]b. Here, both drugs separately and the combination of them reduced the formation of 3D colonies in GOS-3 glioblastoma cells respect to control. In summary, the combination treatment is the best one to treat GOS-3 glioblastoma cells, just looking at the results of the clonogenicity assays.
Figure 1: Colony formation assay (a) and soft agar clonogenic assay (b) in GOS-3 glioblastoma cells. Panob: Panobinostat. Both: Panobinostat and 3-deazaneplanocin-A. **P < 0.01 vs. control, ***P < 0.001 vs. control, •P < 0.05 vs. joint treatment, ••P < 0.01 vs. joint treatment, •••P < 0.001 vs. joint treatment

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3-Deazaneplanocin-A reduces cell migration of GOS-3 glioblastoma cells

After results obtained by the 2D and 3D colony formation assays, cell migration was expected to be affected in GOS-3 cells. A scratching test or wound-healing assay was performed for the evaluation of cell migration [Figure 2]. DZ-Nep-treated cells were the ones which migrated less, followed by panobinostat-treated cells and finally by the cells under the combined treatment. In summary, according to the results of the migration assays, DZ-Nep is better than panobinostat and much better than the combined treatment, to treat GOS-3 glioblastoma cells. Moreover, the combined treatment was even worse than control based on the cell migratory effect, as cells treated with the combination of panobinostat and DZ-Nep migrated more than control cells.
Figure 2: Wound-healing (scratching) assay in GOS-3 glioblastoma cells. Panob: Panobinostat. Both: Panobinostat and 3-deazaneplanocin-A

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Changes in gene expression are induced in GOS-3 glioblastoma cells by the epigenetic treatments

To know the effects of panobinostat, DZ-Nep, and the combined treatments on gene expression, RT-qPCR of several genes [Table 1] and [Figure 3] was performed for all treatments carried out. Genes studied were selected due to their role in apoptosis, genetics, and epigenetics of glioblastoma. BAX and NOXA are proapoptotic genes. EZH2 was analyzed because DZ-Nep is a direct inhibitor of this gene. SUZ12 is, together with EZH2, a component of the PRC2. PHF1 is a PRC2 recruiter. TIMP3 (metastasis inhibitor), VASH1 (angiogenesis inhibitor), and CDH1 (E-cadherin gene) are EZH2 targets. MGMT (DNA methyltransferase crucial for genome stability) and CDKN2A (p16, cell cycle inhibitor) are commonly altered genes in glioblastoma.
Figure 3: Real-time quantitative polymerase chain reaction expression of NOXA, BAX, enhancer of zeste homolog 2, suppressor of zeste 12 homolog, PHF1, vasohibin 1, tissue inhibitor of metalloproteinase 3, CDH1, O(6)-methylguanine-DNA methyltransferase and cyclin-dependent kinase inhibitor 2A genes in GOS-3 glioblastoma cells. Panob: Panobinostat. Both: Panobinostat and 3-deazaneplanocin-A. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control, •P < 0.05 vs. joint treatment, ••P < 0.01 vs. joint treatment, •••P < 0.001 vs. joint treatment

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The expression of both proapoptotic genes studied, NOXA and BAX [Figure 3], was increased with the combined treatment. Panobinostat or DZ-Nep alone was partially efficient, but only to increase BAX expression. Their separate action on NOXA was inefficient. This reaffirms the ability of the combined treatment to induce apoptosis in GOS-3 cells.

The expression study of the three genes related to the PRC2 complex [Figure 3] revealed that DZ-Nep was able to only slightly reduce EZH2. It also reduced PHF1 expression and overexpressed SUZ12. Panobinostat produced opposite effects compared to DZ-Nep (increased EZH2 and PHF1) and also increased SUZ12 although less than DZ-Nep did. The effect of the combined treatment was more visible on PHF1 expression, which was greatly increased respect to individual treatments. SUZ12 was also increased, but less than with individual treatments. Finally, EZH2 slightly increased its expression by the combined treatment. In summary, only DZ-Nep was able to reduce EZH2 and PHF1, while SUZ12 was elevated in any condition (most notoriously in DZ-Nep-treated cells).

Respect to the suppressor genes involved in angiogenesis and metastases [Figure 3], the combined treatment was more efficient than panobinostat or DZ-Nep alone to increase TIMP3 and CDH1 (E-cadherin gene) expression, while panobinostat produced the highest levels of VASH1 expression.

Finally, panobinostat alone could produce the highest levels of CDKN2A expression that were needed to brake the cell cycle and inhibit cell growth. DZ-Nep and the combination treatment could equally decrease MGMT expression [Figure 3] needed for inducing chemosensitivity to temozolomide in the glioblastoma clinical treatment.[37]

To test at the protein level whether DZ-Nep inhibited EZH2 in GOS-3 cells, we performed a western blot with a primary antibody against H3K27 me3 [Figure 4]. Proteins extracted from cells treated with the different compounds (DZ-Nep, panobinostat, and the double treatment) versus untreated control cells were tested. A beta-actin primary antibody was also used as a loading control. Contrary to what was expected, DZ-Nep-treated cells did not show a reduced amount of H3K27 me3 protein versus untreated control cells, which shows that for some reason, DZ-Nep does not seem to act as a proper inhibitor of EZH2 in GOS-3 cells. This is in agreement with the results presented in [Figure 3], in which DZ-Nep slightly inhibited EZH2 mRNA expression. Panobinostat, as expected, and the double treatment showed a similar level of H3K27 me3 protein expression as the untreated control cells did.
Figure 4: Western blot protein expression of H3K27 me3 and beta-actin in GOS-3 glioblastoma cells. Panob: Panobinostat. Both: Panobinostat and 3-deazaneplanocin-A

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  Discussion Top

GOS-3 glioblastoma cells were chosen for the study. Although first considered to be a Grade II/III astrocytoma/oligodendroglioma cell line,[28],[29] GOS-3 was further characterized as a glioblastoma cell line.[30] The drugs used against GOS-3 cells, DZ-Nep and panobinostat, inhibit EZH2 and HDAC, respectively, which are enzymes that play a fundamental role in gene silencing by methylation or deacetylation of histones.

The two assays for testing in vitro cell tumorigenicity, the 2D colony formation assay and the 3D clonogenic assay in soft agar, were significantly altered with the treatments used: the number of colonies formed was highly decreased respect to the nontreated control cells when the combination of DZ-Nep and panobinostat was used [Figure 1]. On the contrary, the combined therapy was less efficient than DZ-Nep alone (or panobinostat alone) in reducing cell migration in the wound-healing assay [Figure 2]. These results show that several other cellular tests have to be employed with different cell lines before taking any result as a concluding one. We should admit that our results are preliminary although the combination of DZ-Nep and panobinostat seems to be the best approach to treat GOS-3 glioblastoma cells; according to the clonogenic results, the same combination produced the worst result in terms of reducing cell migration. On the contrary, if we look at the migration results and choose DZ-Nep as the best candidate to highly reduce cell migration, we realize that DZ-Nep cannot be a true candidate in terms of inhibiting clonogenicity, as it is indeed the worst compound in inhibiting the formation of 2D or 3D colonies in GOS-3 glioblastoma cells.

Ten genes were explored for their level of expression by RT-qPCR after treatments versus nontreated control cells: two pro-apoptotic genes (BAX and NOXA), three PRC2-related genes (EZH2, SUZ12, and PHF1), two inhibitors of metastasis (TIMP3 and CDH1), one inhibitor of angiogenesis (VASH1), one cell cycle inhibitor (CDKN2A), and one gene with a clinical role in neuro-oncology (MGMT).[37]

It has been documented that BAX activation blocks self-renewal and induces apoptosis of human glioblastoma stem cells, together with sensitizing both glioblastoma cells and glioblastoma stem cells to temozolomide, which reveals that BAX activation in glioblastoma might be a promising therapeutic tool against the stem cell reservoir.[38] In our work, we demonstrated that expression of both proapoptotic genes studied, NOXA and BAX [Figure 3], was increased with the combined treatment. It was the best one, compared to panobinostat or DZ-Nep alone, to induce the expression of both genes.

Respect to the three genes related to the PRC2, EZH2 is the catalytic domain that trimethylates H3K27. EZH2 has been documented to have pro-oncogenic activity in various types of tumors, such as glioblastoma,[39],[40] sarcoma,[41] breast,[25] prostate,[24],[32] colorectal,[42] and bladder cancer.[43] However, EZH2 has a context-dependent role in different cancers, as an oncogene or tumor suppressor. Experiments in a mouse model of MYC-associated group 3 medulloblastoma have shown that engineered deletions of EZH2 by gene editing nucleases accelerated tumorigenesis, whereas EZH2 reexpression reversed histone modifications and slowed tumor progression.[44] This might show that EZH2 somehow downregulates MYC in medulloblastoma. But MYCN upregulates EZH2 in neuroblastomas,[45] which supports testing for EZH2 inhibitors in patients with MYCN-amplified neuroblastoma. Moreover, E2F1 transcription factor bound the proximal EZH2 and SUZ12 promoter to activate transcription in bladder cancer, suggesting that E2F1 and its downstream effectors, EZH2 and SUZ12, could be important mediators for cancer progression.[43] EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, against glioma stem cells, suggesting strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.[46] Other strategies for EZH2 targeting have been published elsewhere.[22],[23],[27]

SUZ12 is one of the three components of the PRC2 (SUZ12, EZH2, and EED). It is usually highly expressed in cancer cells,[39],[40],[41],[42] and considered as an oncogene. SUZ12 expression was significantly increased in gastric cancer tissues,[47] suggesting that its upregulation may be a negative prognostic factor. Knockdown of SUZ12 expression impaired cell proliferation and invasion in vitro, leading to the inhibition of metastasis in vivo.[47] Targeting SUZ12 by RNA interference inhibits the invasion of gastric carcinoma cells and the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9.[48] MicroRNA-105 inhibits human glioma cell malignancy by directly targeting SUZ12.[49] MicroRNA-128 coordinately targets polycomb repressor complexes in glioma stem cells.[50]

On the contrary to the conception of SUZ12 as an oncogene, there are studies that reinforce the idea of SUZ12 behaving as a tumor-suppressor gene.[51],[52],[53],[54],[55] It has been demonstrated that PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors.[54],[55] Upregulated miR-489 decreased SUZ12 mRNA expression level, increased E-cadherin, and decreased N-cadherin and vimentin protein levels, suppressing cell invasion.[51] Moreover, the JAZF1–SUZ12 fusion protein[51] disrupts PRC2 complexes and impairs chromatin repression during human endometrial stromal tumorigenesis.[53]

Polycomb-like proteins, such as PHF1, MTF2, and PHF19, are PRC2-associated factors that form subcomplexes with PRC2 core components and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci.[56]

Our results on the expression of the two genes of the PRC2 (EZH2 and SUZ12) and of PHF1, the PRC2 recruiter gene [Figure 3], revealed that DZ-Nep was able to slightly reduce EZH2. It also reduced PHF1 expression and overexpressed SUZ12. Compared to control cells, we might infer, in light of the results induced by DZ-Nep, that EZH2 and PHF1 behave as oncogenes, while SUZ12 seems to be a tumor suppressor gene in GOS-3 glioblastoma cells, as suggested elsewhere.[51],[52],[53],[54],[55] Panobinostat and the combined treatment increased the level of the three markers, which is not desired for EZH2 and PHF1.

TIMP3 is an inhibitor of metastasis.[57],[58],[59],[60] Therefore, its expression should be guaranteed. Overexpression of TIMP3 inhibits tumor angiogenesis.[59] Silencing of TIMP3 would lead to cell invasion and metastasis:[57],[58],[60] KDM1A (a lysine demethylase) promotes tumor cell invasion by silencing TIMP3 in nonsmall cell lung cancer cells;[58] miR-21, by inhibiting TIMP3, promotes cell invasion in renal [57] and cervical[60] cancer.

VASH1 (an inhibitor of angiogenesis) has conceptually been considered as a tumor-suppressor gene.[61] Other studies define it as a pro-oncogenic factor in different cancers such as gastric,[62] bladder,[63] renal,[64] and colorectal,[65] and in squamous cell carcinoma.[66]

CDH1 codes for E-cadherin, which has been considered to act both as an oncogene[67] and as a tumor-suppressor gene [68],[69],[70],[71] in cancer development. Thus, unlike in epithelial tissues, E-cadherin expression in glioma correlated with an unfavorable clinical outcome,[67] and shRNA knockdown of E-cadherin in SF767 glioma cells resulted in decreased proliferation and migration in vitro.[67] A high expression of transforming growth factor-beta 1 and a low expression of E-cadherin both participate in the incidence, development, and migration of glioma.[71] Respect to the molecular mechanism for E-cadherin participation, knockdown of ILK inhibited glioma cell migration, invasion, and proliferation through upregulation of E-cadherin and downregulation of cyclin D1.[69] Moreover, GLI2 overexpression was associated with decreased E-cadherin protein levels; increased expression of SNAIL, matrix metalloproteinase 2, and integrin beta1; and increased cell invasion in 3D organotypic cultures.[70] Finally, E-and N-cadherin, as representative epithelial–mesenchymal transition (EMT) markers, have limited prognostic value in glioma.[68]

Our results on the expression of the three genes related to angiogenesis and metastasis (TIMP3, VASH1, and CDH1) [Figure 3] showed that the combined treatment was more efficient than panobinostat or DZ-Nep alone to increase TIMP3 and CDH1 (E-cadherin gene) expression, while panobinostat produced the highest levels of VASH1 expression. Therefore, the combined treatment, respect to the expression of these genes, should be the one to be selected.

CDKN2 codes for p16 tumor-suppressor protein. CDKN2A decreased expression is a marker of poor prognosis in malignant high-grade glioma.[72] Epigenetic studies on the role of histone methylases in the development of brainstem pediatric glioma have shown that H3.3K27M mutation represses CDKN2A expression in a genetic mouse model, which constitutes a promising therapeutic avenue.[73]

Silencing of the MGMT promoter is thought to induce chemosensitivity to temozolomide, and testing for methylation may allow for patient stratification.[37] In a subset of glioblastomas, the MGMT promoter is methylated, impairing the repair mechanism and conferring chemosensitivity, which leads scientists and clinicians to fight for treatment considerations for MGMT-unmethylated glioblastoma.[74] On that line of research, miR-198 overexpression has been demonstrated to prevent protein translation of MGMT and enhance temozolomide sensitivity in glioblastoma.[75]

Our results on CDKN2A and MGMT expression after treatments with panobinostat, DZ-Nep, or both, versus nontreated GOS-3 glioblastoma cell lines, showed that panobinostat alone could produce the highest levels of CDKN2A expression, while DZ-Nep and the combination treatment could equally decrease MGMT expression [Figure 3]. Therefore, panobinostat would be the preferred treatment to allow for breaking cell cycle through CDKN2A (p16) elevation and inducing chemosensitivity to temozolomide in glioblastoma cells through MGMT inhibition of expression.

After having treated GOS-3 glioblastoma cells with panobinostat, DZ-Nep, or both, and having compared cell behavior and gene expression in treated cells versus nontreated control cells, we can reach some conclusions [Figure 5]. Panobinostat alone or the combination of panobinostat plus DZ-Nep, which would theoretically induce the expression of target genes due to demethylation and further acetylation of H3K27, seem to be the two main conditions for a proper treatment that may inhibit clonogenicity, metastasis, angiogenesis, EMT, and entry in the S phase of the cell cycle and that may induce apoptosis in GOS-3 glioblastoma cells. On the contrary, DZ-Nep would be the best choice for inhibiting cell migration (single treatment) and MGMT expression (DZ-Nep or double treatment). To reduce the expression of EZH2 and of its recruiter, PHF1, then demethylating H3K27, DZ-Nep should be used; however, SUV12, a component of the PRC2, would then increase, according to our results, pointing at it behaving as a tumor-suppressor gene, notion already presented by others.[51],[52],[53],[54],[55] Finally, we might conclude that a combination of DZ-Nep and temozolomide might be a good choice against GOS-3 glioblastoma cells as DZ-Nep would decrease MGMT expression, thus chemosensitizing GOS-3 cells to temozolomide.
Figure 5: Strategy of epigenetic treatment of GOS-3 glioblastoma cells and results obtained

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Previous studies from our group point that panobinostat and temozolomide combination [36],[76] or panobinostat and DZ-Nep[35] combination might be more efficient against glioblastoma cells than just temozolomide. Our research highlights the importance of pharmacologic epigenetic modulation in glioblastoma, as a potential adjuvant therapy to be used with temozolomide. Nevertheless, more experiments with more markers studied in different cell lines and tumors, not only in vitro but also under in vivo conditions, such as xenotransplantation of treated cells to nude mice, will help elucidate the best strategies to help combat glioblastoma.

Financial support and sponsorship

Financial support for this work was provided by a grant from the Fundación Universidad de Navarra, Pamplona, Spain. J.R. and A.U. received predoctoral fellowships from the Asociación de Amigos de la Universidad de Navarra, Pamplona, Spain.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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