|Year : 2018 | Volume
| Issue : 1 | Page : 22-26
In vitro therapy against glioblastoma cells by 3-Dezaneplanocin-A, panobinostat, and temozolomide
Javier de la Rosa1, Alejandro Urdiciain1, Bárbara Meléndez2, Juan A Rey3, Miguel A Idoate4, Javier S Castresana1
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 Web Publication||28-Feb-2018|
Javier S Castresana
Department of Biochemistry and Genetics, University of Navarra School of Sciences, Irunlarrea 1, Pamplona 31008
Source of Support: None, Conflict of Interest: None
Background: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current treatment against this tumor consists of maximal surgical resection without threatening the patient's life, followed by a treatment with temozolomide, with or without combined radiotherapy. GBM is resistant to the conventional antitumor therapies, so in this research, we tried to inhibit tumor growth with the combination of three drugs: (1) panobinostat, an inhibitor of histone deacetylases, (2) 3-Dezaneplanocin-A (DZNep), an inhibitor of EZH2, a protein which belongs to the polycomb repressor complex 2, acting as a histone methylase, and (3) temozolomide, an alkylating agent. Methods: The T98G GBM commercial cell line was used. Cells were exposed to single treatments of the drugs and to the three possible combinations among them. Soon after, two-dimensional (2D) and 3D clonogenic assays were assessed for in vitro tumorigenicity testing. Real-time quantitative polymerase chain reaction of 2 proapoptotic genes (BAX and NOXA) and 2 antiapoptotic genes (BCL2 and BCL-XL) was also assessed. Results: The panobinostat and temozolomide combination produced a positive effect against T98G glioblastoma cells by reducing soft agar colony formation, by inducing high expression levels of NOXA, and by reducing BCL-XL expression. Equally, the panobinostat and DZNep combination produced a positive effect against T98G glioblastoma cells by reducing colony formation in adherent conditions and by inducing high expression levels of BAX. Finally, temozolomide alone was the most efficient drug for decreasing BCL2 expression. Conclusion: Panobinostat and temozolomide combination or panobinostat and DZNep combination might be more efficient against glioblastoma cells than just temozolomide.
Keywords: Brain tumors, epigenetics, EZH2, histone deacetylase, histone methylase
|How to cite this article:|
de la Rosa J, Urdiciain A, Meléndez B, Rey JA, Idoate MA, Castresana JS. In vitro therapy against glioblastoma cells by 3-Dezaneplanocin-A, panobinostat, and temozolomide. Glioma 2018;1:22-6
|How to cite this URL:|
de la Rosa J, Urdiciain A, Meléndez B, Rey JA, Idoate MA, Castresana JS. In vitro therapy against glioblastoma cells by 3-Dezaneplanocin-A, panobinostat, and temozolomide. Glioma [serial online] 2018 [cited 2022 Nov 30];1:22-6. Available from: http://www.jglioma.com/text.asp?2018/1/1/22/226434
Javier de la Rosa and Alejandro Urdiciain have contributed equally.
| Introduction|| |
Glioblastoma multiforme (GBM) is considered to be the most aggressive intracranial malignant tumor,, with 14.7 months median survival after diagnosis. Current treatment consists of surgery, radiotherapy, and temozolomide as the gold standard chemical compound. GBM presents high inter- and intra-tumor heterogeneity , and responds in a heterogenic way to temozolomide, even presenting resistance to temozolomide, which added to the difficulty for most drugs to cross the blood–brain barrier, making GBM one of the most difficult tumors to be clinically treated. Therefore, research efforts to develop efficient therapies against GBM are welcome and required.
One of the possible new approaches to be explored to try to combat GBM is epigenetic regulation of gene expression. On one hand, histone deacetylase (HDAC) inhibitors, such as panobinostat, would inhibit deacetylation of histones and would therefore procure a relaxed-chromatin status of adjacent genes, which would keep their expression on. Panobinostat has been shown to break some pathways which play an important role in cancer development  and is beginning to be used against GBM.,,,,,
On the other hand, histone methylases like EZH2 of the polycomb repressor complex 2,, trimethylate lysine 27 of histone 3, inducing an off status of expression of adjacent genes. If these genes are tumor suppressor genes (TSGs), cancer develops. EZH2 acts, then, as an oncogene and its expression associates to poor prognosis., EZH2 expression should be reduced in tumors. One way to do so is by the use of 3-Dezaneplanocin-A (DZNep), an inhibitor of EZH2, which upregulates TSGs and may avoid or, at least, decrease tumor growth. Abnormal elevated levels of EZH2 appear in breast cancer, and prostate cancer, among other tumors.
Clinical trials of panobinostat have recently been performed. So far, two studies on multiple myeloma have reached Phase III,, suggesting that panobinostat could be a useful addition to conventional treatments  although there was only a modest overall survival benefit after the addition of panobinostat. Three clinical trials have been made on glioblastoma.,, One of them was a Phase II clinical trial  in which panobinostat plus bevacizumab did not significantly improve progression-free survival rate. The other two , corresponded to Phase I clinical trials. DZNep has not been included in clinical trials so far. However, the in vitro testing of DZNep in cancer cells makes of it an interesting therapeutic candidate.,,,
In our work, we have treated T98G GBM cells with temozolomide, panobinostat, and DZNep, in simple treatments or in double combinations. Our results, in terms of changes in cell clonogenicity and in expression of proapoptotic and antiapoptotic genes after treatments, show that, in general, some of the double treatments might be more efficient than temozolomide alone when trying to combat GBM cells.
| Materials and Methods|| |
The present project has been approved by the Ethics Committee of the University of Navarra with reference number CEI0502012. T98G cell line was cultured in RPMI L-GlutaMAX medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Invitrogen Corporation, Carlsbad, CA, USA). It was maintained in an incubator at 37°C in an atmosphere with 5% CO2 to grow the cells.
When confluence was around 80%, cells were harvested for subculture. The same medium was added to the drugs used in this study. Used concentrations for the drugs were: DZNep, 5 μM; panobinostat, 20 nM; and temozolomide, 200 nM. Drugs were added as a simple treatment or in a double combination for a period of 3 days. Medium with the treatment was changed every day and contained the same quantity of DMSO, always with a concentration below 0.1%.
Colony formation assay
To study the clonogenic potential of cells in attachment-dependent conditions, 300 T98G cells were cultured in 6-well plates for 14 days. Then, cells were fixed with paraformaldehyde 4% for 30 min and stained with crystal violet (Sigma Aldrich Corporation, St Louis, MO, USA) for 15 min. Resulting colonies were counted using a Colony 560 Counter (Suntex). The experiment was repeated three times.
Soft agar clonogenic assay
To study the clonogenic potential of cells in attachment-independent conditions, T98G cells were cultured in agarose in a 6-well plate. Before that, 2 mL of agarose 0.5% (Cat No. 8016, PRONADISA, Laboratorios Conda, Torrejón de Ardoz, Madrid, Spain) and DMEM (Sigma Aldrich Corporation, St Louis, MO, USA) were added. Once this first layer was solidified, 20,000 cells contained in 2 mL of agarose 0.2% and DMEM were added onto the first agar layer. When the top layer got solidified, 2 mL of RMPI-GlutaMAX medium supplemented with FBS 10% were added and changed every 3 days. Four weeks later, the medium was discarded, and the colonies were stained with 250 μL of crystal violet 1% overnight. Samples were washed with H2O to improve visualization of the colonies. To count the colonies, 5 photos per well were taken. The different drug conditions were cultured three times for each condition of each cell line.
mRNA was extracted using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) from the pellets after treating cells for 72 h with the different drugs and their respective combinations. mRNA quantification was performed with the Nanodrop system.
To obtain cDNA from mRNA, a retrotranscription was conducted from 2 μg of mRNA, 250 μg of random primers, and 2 μL of dNTPs (stock at 5 μM), in a final volume of 12 μL of RNAse-free water. The mix was incubated for 5 min at 65°C, and then, 4 μL of buffer 5X and 2 μL of DTT were added. Two minutes later, 1 μL of the enzyme SuperScriptTM II reverse transcriptase (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) was added, and the mix was incubated for 10 min at 25°C, 50 min at 42°C, and 15 min at 70°C. The resulting cDNA was diluted 1/5 in H2O and was kept at −20°C.
Real-time quantitative polymerase chain reaction
Gene expression was measured by real-time (RT) quantitative polymerase chain reaction (PCR). Analysis and design of the pair of primers were carried out using Primer3 software (http://primer3.sourceforge.net/) in order to obtain the product length, %GC, Tm, and other characteristics we wished. These primers were paired to the sequences from RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq/) and UCSC Genome Browser (http://genome.ucsc.edu/). An IQ5 multicolor RT PCR detection system (Bio-Rad, Hercules, CA, USA) was used for the PCR reactions. 1.25 μL of cDNA, 12.5 μL of Sybr Green (Cat No. #170-8882, Bio-Rad, Hercules, CA, USA), 0.5 μL of forward and reverse primers, and 10.25 μL of miliQ water were added to each well. The samples followed the following protocol: a denaturalization cycle at 95°C for 10 min, followed by 45 cycles of amplification consisting of 30 s at 95°C, 30 s at hybridization temperature (annealing) of each gene, and 30 s at 72°C. Then, the analysis of the melting temperature curve from 70°C to 90°C was made every 0.5°C. The transcripts of the gene were normalized with the transcripts of the housekeeping gene of each sample (GAPDH). Quantification and normalization of genes were assessed by the 2ΔΔC method. For each sample, 3 wells were measured.
Statistical analysis was performed using GraphPad Prism 7. An ANOVA study was chosen to compare the results from the 7 different groups assayed. The post hoc test chosen was Tukey's. The results represent mean ± standard deviation (SD). Differences were considered statistically significant if the P < 0.05 (*), 0.01 (**) and 0.001 (***).
| Results|| |
Colony formation is inhibited with double treatments
Inhibition of colony formation [Figure 1]A was more remarkable when double treatments were added to the cells compared to control. The combination of DZNep with panobinostat was the treatment with the highest potential to inhibit colony formation in T98G cells, in which no colonies were counted (P< 0.001) [Figure 1]A. The other two treatment combinations presented statistical significance compared to control, as well (P< 0.001). The single treatments revealed as less efficient than the combinations in relation to colony inhibition.
|Figure 1: (A) Colony formation assay in treated T98G cells. (B) Soft agar clonogenic assay in treated T98G cells. Panob: Panobinostat, Temoz: Temozolomide. Error bars: n = 3, mean ± standard deviation, *P < 0.05 and ***P < 0.001 vs. control, d vs. 3-Dezaneplanocin-A, π vs. panobinostat|
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Colony formation in soft agar is more efficiently inhibited with double treatments
In this assay [Figure 1]B, the most remarkable treatment was the combination of panobinostat with temozolomide (P< 0.001) [Figure 1]B, obtaining a significant response with respect to control or to panobinostat alone. The other two combinations showed better results than control or DZNep alone (P< 0.001). Single treatments presented a notable inhibitory effect with respect to the control condition (P< 0.001).
Real-time quantitative polymerase chain reaction
The mRNA expression level of BAX proapoptotic gene was upregulated when double treatments were added. The most remarkable treatment corresponded to the DZNep and panobinostat combination, which significantly increased BAX expression (P< 0.001) with respect to control, DZNep alone, or panobinostat alone [Figure 2]A. The other two combinations were more efficient than the drugs alone but did not show a statistically significant response with respect to control.
|Figure 2: (A) BAX expression in T98G. (B) NOXA expression in T98G. (C) BCL-2 expression in T98G. (D) BCL-XL expression in T98G. Panob: Panobinostat, Temoz: Temozolomide. Error bars: n =, mean ± standard deviation, *P < 0.05 and ***P < 0.001 vs. control, d vs. 3-Dezaneplanocin-A, π vs. panobinostat, t vs. temozolomide by Tukey test|
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The best effect of the different drug combinations in the study of mRNA expression of the proapoptotic NOXA gene was found when the panobinostat and temozolomide double treatment was used, with a 3-fold increase in NOXA expression with respect to the control condition (P< 0.001) [Figure 2]B. The double combination of DZNep with panobinostat also significantly increased NOXA expression.
In the study of the antiapoptotic gene BCL2, we can highlight that the panobinostat plus temozolomide double treatment significantly increased BCL2 mRNA expression with respect to control, panobinostat alone, or temozolomide alone (P< 0.001) [Figure 2]C. DZNep alone and temozolomide alone significantly decreased BCL2 expression levels with respect to control, being any of them separately, and not the combined treatments, the best options for decreasing BCL2 antiapoptotic agent.
BCL-XL antiapoptotic gene was decreased at levels of no mRNA expression under the panobinostat and temozolomide double treatment, in a significant manner with respect to control, panobinostat alone, or temozolomide alone (P< 0.001) [Figure 2]D. DZNep and temozolomide double treatment was the second option to significantly decrease BCL-XL with respect to control and to DZNep alone. Finally, panobinostat alone and temozolomide alone also efficiently inhibited this antiapoptotic gene.
| Discussion|| |
Current treatment for GBM makes use of maximal surgical resection without threatening the patient's life, followed by a treatment with temozolomide, with or without combined radiotherapy; however, the median survival for these patients is 14.7 months. This highlights the importance of searching for an effective therapy against this tumor.
The aim of this study was to analyze if panobinostat and DZNep, both epigenetic drugs, might behave as adjuvant antineoplastic agents of temozolomide, currently used in the clinic for the treatment of glioblastoma. Therefore, the three drugs were experimented alone or in double combinations to treat T98G glioblastoma cells. The efficacy of the treatments was assayed by two kinds of in vitro clonogenic assays: in adherent and in nonadherent (in soft agar) conditions. Moreover, the expression of two proapoptotic genes (BAX and NOXA) and of two antiapoptotic genes (BCL2 and BCL-XL) was tested in the six treatment options and in the controls.
Our evidence supports that double treatments in which panobinostat was included were more efficient in terms of decreasing colony formation in adherent conditions (panobinostat and DZNep) or in soft agar (panobinostat and temozolomide). With respect to the proapoptotic genes, BAX expression was especially increased with the panobinostat and DZNep combination, while the highest efficacy for getting NOXA high expression levels was conducted by the panobinostat and temozolomide combination. Finally, the highest reduction of BCL-XL antiapoptotic gene expression was produced by the panobinostat and temozolomide combination as well, while for BCL2, this happened by temozolomide alone, as the panobinostat and temozolomide combination induced the opposite effect, a big elevation of BCL2 expression.
We might then admit that the panobinostat and temozolomide combination produced a positive effect against T98G glioblastoma cells by reducing soft agar colony formation, by inducing high expression levels of NOXA, and by reducing BCL-XL expression. Equally, the panobinostat and DZNep combination produced a positive effect against T98G glioblastoma cells by reducing colony formation in adherent conditions and by inducing high expression levels of BAX. Finally, temozolomide alone was the most efficient drug for decreasing BCL2 expression.
The positive effect of panobinostat against T98G glioblastoma cells might be due to the induction of cycle arrest and tumor death that HDAC inhibitors can produce., Second, tumors with overexpression of HDAC6 have a greater resistance to temozolomide. Third, we should take into account that panobinostat can possibly acts as an inhibitor of EZH2, which adds a new function to its known function – inhibitor of HDAC – and that might explain the powerful effect of panobinostat combined with the other two drugs: DZNep and temozolomide.
More studies including phenotypically diverse cell lines and primary cultures derived from tumor samples should be performed to test whether these preliminary data on the efficacy of panobinostat against glioblastoma are conclusive.
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.
| References|| |
Jovčevska I, Kočevar N, Komel R. Glioma and glioblastoma-how much do we (not) know? Mol Clin Oncol 2013;1:935-41.
Hamza MA, Gilbert M. Targeted therapy in gliomas. Curr Oncol Rep 2014;16:379.
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al.
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N
Engl J Med 2005;352:987-96.
Meyer M, Reimand J, Lan X, Head R, Zhu X, Kushida M, et al.
Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Proc Natl Acad Sci U S A 2015;112:851-6.
Pardridge WM. The blood-brain barrier: Bottleneck in brain drug development. NeuroRx 2005;2:3-14.
Lo HW. EGFR-targeted therapy in malignant glioma: Novel aspects and mechanisms of drug resistance. Curr Mol Pharmacol 2010;3:37-52.
Atadja P. Development of the pan-DAC inhibitor panobinostat (LBH589): Successes and challenges. Cancer Lett 2009;280:233-41.
Berghauser Pont LM, Kleijn A, Kloezeman JJ, van den Bossche W, Kaufmann JK, de Vrij J, et al.
The HDAC inhibitors scriptaid and LBH589 combined with the oncolytic virus delta24-RGD exert enhanced anti-tumor efficacy in patient-derived glioblastoma cells. PLoS One 2015;10:e0127058.
Lee EQ, Reardon DA, Schiff D, Drappatz J, Muzikansky A, Grimm SA, et al.
Phase II study of panobinostat in combination with bevacizumab for recurrent glioblastoma and anaplastic glioma. Neuro Oncol 2015;17:862-7.
Pont LM, Naipal K, Kloezeman JJ, Venkatesan S, van den Bent M, van Gent DC, et al.
DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells. Cancer Lett 2015;356:525-35.
Singleton WG, Collins AM, Bienemann AS, Killick-Cole CL, Haynes HR, Asby DJ, et al.
Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model. Int J Nanomedicine 2017;12:1385-99.
Yao ZG, Li WH, Hua F, Cheng HX, Zhao MQ, Sun XC, et al.
LBH589 inhibits glioblastoma growth and angiogenesis through suppression of HIF-1α expression. J Neuropathol Exp Neurol 2017;76:1000-7.
Yu C, Friday BB, Yang L, Atadja P, Wigle D, Sarkaria J, et al.
Mitochondrial bax translocation partially mediates synergistic cytotoxicity between histone deacetylase inhibitors and proteasome inhibitors in glioma cells. Neuro Oncol 2008;10:309-19.
Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. Genome regulation by polycomb and trithorax proteins. Cell 2007;128:735-45.
Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, et al.
The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002;419:624-9.
Collett K, Eide GE, Arnes J, Stefansson IM, Eide J, Braaten A, et al.
Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer. Clin Cancer Res 2006;12:1168-74.
Goodman RH, Smolik S. CBP/p300 in cell growth, transformation, and development. Genes Dev 2000;14:1553-77.
Kim KH, Roberts CW. Targeting EZH2 in cancer. Nat Med 2016;22:128-34.
San-Miguel JF, Hungria VT, Yoon SS, Beksac M, Dimopoulos MA, Elghandour A, et al.
Panobinostat plus bortezomib and dexamethasone versus placebo plus bortezomib and dexamethasone in patients with relapsed or relapsed and refractory multiple myeloma: A multicentre, randomised, double-blind phase 3 trial. Lancet Oncol 2014;15:1195-206.
San-Miguel JF, Hungria VT, Yoon SS, Beksac M, Dimopoulos MA, Elghandour A, et al.
Overall survival of patients with relapsed multiple myeloma treated with panobinostat or placebo plus bortezomib and dexamethasone (the PANORAMA 1 trial): A randomised, placebo-controlled, phase 3 trial. Lancet Haematol 2016;3:e506-15.
Drappatz J, Lee EQ, Hammond S, Grimm SA, Norden AD, Beroukhim R, et al.
Phase I study of panobinostat in combination with bevacizumab for recurrent high-grade glioma. J Neurooncol 2012;107:133-8.
Shi W, Palmer JD, Werner-Wasik M, Andrews DW, Evans JJ, Glass J, et al.
Phase I trial of panobinostat and fractionated stereotactic re-irradiation therapy for recurrent high grade gliomas. J Neurooncol 2016;127:535-9.
Choudhury SR, Balasubramanian S, Chew YC, Han B, Marquez VE, Eckert RL, et al.
(-)-epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells. Carcinogenesis 2011;32:1525-32.
Lee JK, Kim KC. DZNep, inhibitor of S-adenosylhomocysteine hydrolase, down-regulates expression of SETDB1 H3K9me3 HMTase in human lung cancer cells. Biochem Biophys Res Commun 2013;438:647-52.
Uchiyama N, Tanaka Y, Kawamoto T. Aristeromycin and DZNeP cause growth inhibition of prostate cancer via induction of mir-26a. Eur J Pharmacol 2017;812:138-46.
Zhou J, Bi C, Cheong LL, Mahara S, Liu SC, Tay KG, et al.
The histone methyltransferase inhibitor, DZNep, up-regulates TXNIP, increases ROS production, and targets leukemia cells in AML. Blood 2011;118:2830-9.
Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK, et al.
Histone deacetylases and cancer: Causes and therapies. Nat Rev Cancer 2001;1:194-202.
Kim IA, No M, Lee JM, Shin JH, Oh JS, Choi EJ, et al.
Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: Potential role of histone deacetylase 6. Radiother Oncol 2009;92:125-32.
Li ZY, Zhang C, Zhang Y, Chen L, Chen BD, Li QZ, et al.
Anovel HDAC6 inhibitor tubastatin A: Controls HDAC6-p97/VCP-mediated ubiquitination-autophagy turnover and reverses temozolomide-induced ER stress-tolerance in GBM cells. Cancer Lett 2017;391:89-99.
Fiskus W, Pranpat M, Balasis M, Herger B, Rao R, Chinnaiyan A, et al.
Histone deacetylase inhibitors deplete enhancer of zeste 2 and associated polycomb repressive complex 2 proteins in human acute leukemia cells. Mol Cancer Ther 2006;5:3096-104.
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