Abstract

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Background: Up-regulation of energy metabolism is a key feature of proliferating cells from cancers and leukemias. Thus, targeting of metabolic pathways represents a therapeutic challenge involving both dietary and drug-mediated strategies. The aim of this study was to evaluate the impact of the DNA-methyltransferase-inhibitor 5-desoxy-2-azacytidine (Decitabine, DAC) and the histone-deacetylase-inhibitor suberoyl anilide hydroxamic acid (SAHA, Vorinostat) on malignancy-associated metabolic pathways.

Methods: HL60 promyelocytic cells and KG1 myeloblastic leukemia cells were treated with DAC or SAHA for 3 days. RNA was isolated and gene expression was analyzed using Affymetrix human gene 1.0 arrays in comparison with published databases from normal CD34 positive hematopoietic progenitors. Pathvisio software tool was used for comparative quantification of gene-expression patterns from metabolic pathways based on established data banks such as  KEGG and Wikipathways.

Results: A comparison of both leukemia cell lines to normal CD34⁺ cells indicated malignancy-associated stimulation of energy metabolism. Global evaluation on pathways demonstrated a significant drug-mediated downregulation of the intra-mitochondrial pathways, namely the tri-carbonic acid cycle and beta oxidation and to a lesser extent also cytoplasmic glycolysis.

Conclusion: The observation that the KG1 cell line reacted more sensitive to demethylating drugs than the HL60 cell line, where drug treatment selected for survival of differentiated cells, may support application of DAC and SAHA to leukemias with an undifferentiated phenotype but also with the potential to induce differentiation. Efficient targeting of intra-mitochondrial pathways appears to be associated with multiple pro-apoptotic  activities of these drugs.

Introduction

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The main goals of epigenetic therapy include a  selective killing of neoplastic (stem) cells and prevent relapse of malignancy by restoration of a normal DNA methylation pattern in the targeted tissue. Treatment of cancer cells with demethylating agents, such as desoxy- azacytidine    (DAC), inhibits DNA methyltransferases (DNMTs) and decreases DNA methylation, leading to reactivation of gene expression, e.g. of so-called tumor suppressor genes. Azacytidine (5-azacytidine, Vidaza®) and desoxy-azacytidine (DAC, Decitabine, Dacogen®) have been approved as treatments for myelodysplastic syndrome and leukemia (Gurion et al., 2009; Hollenbach et al., 2010; Kuendgen et al., 2011; Morgan et al., 2006).

Histone deacetylase (HDAC) inhibitors, such as suberoyl anilide hydroxamic acid (SAHA, Vorinostat), can induce differentiation, cell cycle arrest and apoptosis (Lee et al., 2009; New et al., 2012). The specific mechanisms explaining these effects are still a matter of research (New et al., 2012), but one possible explanation could be that accumulation of hyperacetylated histones results in activation of tumor suppressor genes and repression of oncogenes. SAHA has been approved for treatment of cutaneous T-cell lymphoma (CTCL) (Zolinza^®). It inhibits HDACs by binding to a zinc ion in the catalytic domain of the enzyme and in doing so preventing the deacetylation of histones and with it gene inactivation (Rountree et al., 2001).  A possible mode of action is shown in Figure 1.

Epigenetically active drugs, such as DAC and SAHA, which were initially designed for re-activation of so-called tumor suppressor genes, regulating cell differentiation and cell death in malignancies, have been shown to affect biochemical pathways. As these processes are also tightly linked to energy metabolism, the aim of this study  was to evaluate the expression signature of the three main metabolic pathways, glycolysis, β-oxidation and TCA cycle, in the KG1 and HL60 leukemia cell lines.

HDAC inhibitors have been proven effective in reversing the metabolic changes in cancer cells as they down-regulate the glucose transporters (GLUT1 and SLC2A1) as well as hexokinase 1 (HK1) and promote apoptosis and growth arrest (Wardell et al., 2009) by cell cycle attenuation in G0/G1 (Tiffon et al., 2011). However, it has to be mentioned that drug-induced metabolic changes could also be a result of mitochondrial damage which has been reported both for DNMT-inhibitors and for histone deacetylase inhibitors (Dell'Aversana et al., 2012; Lee et al., 2012; Ruiz-Magana et al., 2012; Shi et al., 2012). Considering the increasing importance of metabolism-epigenome-crosstalk (Hino et al., 2013; Stefanska et al., 2012), the aim of this study was to evaluate the effect of Decitabine and Vorinostat on key pathways of energy metabolism.

Methods

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Cell culture

The HL60 cells, which were obtained from the American Type Culture Collection, ATCC, Manassas, VA, were seeded in culture flasks in RPMI 1640 (Roswell Park Memorial Institute medium; medium used for growing human lymphoid cells) containing 10 % fetal calf serum (FCS) in 5 % CO2 at a density of 300.000 cells/ml medium. The cells were incubated at 37 °C with either DAC (5 µM) or SAHA for 72 hours (2 µM) in comparison to untreated controls.

The KG1 cells, which were also obtained from the American Type Culture Collection, ATCC, Manassas, VA, were seeded in petri dishes (8 cm in diameter) in RPMI 1640 supplemented with 5 % glutamine, 10 % FCS, 100 units/ml penicillin G sodium and 100 g/ml streptomycin sulphate at a density of 300.000 cells/ml medium. The cells were incubated at 37 °C with either DAC or SAHA for 72 hours in comparison to untreated controls.

Evaluation of drug modulated inhibition of cell multiplication

To estimate the cell multiplication the cells were seeded in 24 wells micro plates at a density of 300.000 cells/ml and cultured for 1, 2 and 3 days with the indicated concentrations (0,5 μM, 1 μM, 2 μM, 4 μM, 8 μM). After the treatment period, the cells were removed from the well and residual adherent cells were detached from the culture plate by treatment with 0.002% pronase E (Roche) and 0.04% EDTA (ethylenediaminetetraacetic acid) in PBS (phosphate buffered saline). Thereafter, all cells of a well were counted with a cell counter (Schärfe,Germany).

RNA isolation for definition of gene expression profiles

Promega Z-3105 RNA Isolation System was used to isolate RNA in order to analyze genome wide mRNA expression with Affymetrix GeneChip Analysis, in order to find out, which genes and associated regulative pathways were affected by treatments with the epigenetic drugs, DAC and SAHA.

Affymetrix gene chip analysis

Performance, analysis and data evaluation for the Affymetrix Arrays (Type Human Gene 1.0 ST Array) were commercially obtained from an internationally certified institution (Kompetenzzentrum für Biofluoreszenz,Regensburg). Pathvisio software was applied for specific analyses of defined pathways from Affymetrix Arrays (Type Human Gene 1.0 ST Array) (van Iersel et al., 2008).

Calculation of Gene Expression Factor

The following formula describing the pathway expression factor was used to describe the regulation of a pathway in a quantitative manner:

PEF= Σlog2signalsTreat -Σlog2signalsContr ninvolved Genes

The Log2 signals represent the intensity of gene expression on the Human Gene 1.0 ST Array (Karlic et al., 2010). Basal expression refers to the log2 of transcript stimulation relative to a standardized panel of unregulated mRNA sequence motifs within the array.

Thus, for comparative analysis of pathway factors it has to be mentioned that “1” means a 2- fold, “2” means a 4-fold, “3” means 8-fold stimulation and further on.

Comparative evaluation of normal CD34+ Cells

In order to interpret the changes in gene expression in the leukemia cell lines and the influence of the epigenetically active drugs, the basal gene expression of undifferentiated hematopoietic CD34+ cells was also evaluated. The GSE3005 gene data set was used for specific analysis via PathVisio. More precisely the sample GSM65673, which consisted of pooled untreated CD34+  cells from 6 healthy donors, kept for 1 hour. The data set GSE3005 was obtained from the date base “GEO DataSet” of theNationalCenterfor Biotechnology Information

(NCBI; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3005).

Gene expression analysis quantitative real time polymerase chain reaction (qRT- PCR)

For comparative analyses cDNA was synthesized from 0.1 μg RNA using the 1^st Strand cDNA Synthesis Kit as described by the supplier (Promega). The obtained cDNA was subjected to PCR amplification with a real-time cycler (Corbett Research). TaqMan gene expression probes & primer-sets (all from Applied Biosystems) in the respective master mix were used for amplification according to the suppliers suggested conditions. For normalization of expression we used the 18S -rRNA TaqMan primer & probe-set in the same reaction vial (Applied Biosystems). Relative quantification of mRNA within the samples was examined using the comparative Ct method:

(DCtuntreated control - DCttreated cells= DDCt; relative quantity = 2-ÄÄCt)  according to a standardized protocol (Livak et al., 2001).

Results

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Dose-effect relations

In order to find the optimal concentration for drug treatment for the leukemia cell lines, the cells were seeded and cultured for 72 hours with increasing concentrations of SAHA as well as of DAC.

Figures 2A-2D show the dose dependent diminution of cell multiplication of the KG1 cell line after treatment with DAC and SAHA. 0.5 μM of DAC and 0.5 μM of SAHA reduced the proliferation rate to about 40% and were, therefore, chosen for the experiments with the KG1 cell line. Figures 2C and 2D illustrate the dose dependent attenuation of cell multiplication of the HL60 cell line after treatment with DAC and SAHA. Thus, for mRNA-expression studies  with the KG1 cell line 0.5 μM of DAC and 0.5 μM of SAHA were selected, whereas for HL60 5 µM DAC or 1 µM SAHA had to be applied.

Comparative analysis of basal gene expression of the genes of the three main metabolic pathways in healthy CD34+ cells and in untreated leukemia cell lines KG1 and HL60

In order to interpret the changes in gene expression in the leukemia cell lines and the influence of the epigenetically active drugs even more accurate, the basal gene expression of the three main metabolic pathways, glycolysis, fatty acid β-oxidation and TCA cycle, in undifferentiated hematopoietic cells, CD34+ cells, as well as the basal gene expression of the untreated KG1 and HL60 cell lines was also analyzed (Suppl Table 1).

In general, it becomes obvious, especially when looking at the colored columns, that most glycolysis-associated genes (except SLC2A3 and SLC2A5 solute carrier family 2 (facilitated glucose transporter), members 3 and 5; HK1 hexokinase 1; PFKM phosphofructokinase, muscle, PFKP phosphofructokinase, platelet, ALDOA and ALDOC, aldolases A and C; ENO2 (enolase 2) and LDHC lactate dehydrogenase C) were higher expressed in the leukemia cell lines as compared to the CD34+ cells. The gene expression factor was calculated in order to get a feeling for the general character of the metabolic state of the leukemia cells. The gene expression factor of the CD34+ cells was6.06,of the KG1 cell line8.04and of the HL60 cell line 7.69.

The gene expression factor of the differences in basal gene expression of the KG1 cell line was 1.99 and of the HL60 cell line 1.63.

All genes related to ß-oxidation were higher expressed in the leukemia cell lines compared to the CD34+ cells. Genes, which were remarkably higher expressed, were ACSL3 in the KG1 cell line, and ACSL4 (acyl-CoA synthetases long-chain family members 3 and 4) and CPT1A (carnitine palmitoyltransferase 1A) in both cell lines.

The gene expression factor, describing the character of the metabolic state of the leukemia cells, of the CD34+ cells was6.51,of the KG1 cell line 8.67 and of the HL60 cell line8.13.The gene expression factor of the differences in basal gene expression of the KG1 cell line was2.17and of the HL60 cell line1.36,respectively.

The genes of the TCA cycle, as well as those related to β-oxidation, were all higher expressed in the leukemia cell lines compared to the CD34+ cells. Genes, which were expressed the highest were DLAT(dihydrolipoamide S-acetyltransferase, a component of pyruvate dehydrogenase complex), IDH3A (isocitrate dehydrogenase 3 (NAD+) alpha), SUCLG2 (succinate-CoA ligase, GDP-forming, beta subunit), SUCLA2 (succinate-CoA ligase, ADP-forming, beta subunit) and MDH2 (malate dehydrogenase 2),  in both cell lines.

The gene expression factor of the CD34+ cells was7.15,of the KG1 cell line 9.65 and of the HL60 cell line9.22.The gene expression factor of the differences in basal gene expression of the KG1 cell line was2.49and of the HL60 cell line2.06;as this refers to log2 values, this means a more than 4-fold higher expression in the cell lines as compared to normal CD34+ cells.

Overview of the three main metabolic pathways, glycolysis, β-oxidation and TCA cycle, targeted by the epigenetically active drugs DAC and SAHA in the leukemia cell lines KG1 and HL60

In the light of recent publications, taking the Warburg effect, caloric restriction and cell senescence into account, understanding the changes in metabolic processes of cancers becomes more and more important. Therefore the three main metabolic

pathways – glycolysis (supplementary Figure 1), fatty acid β- oxidation (supplementary Figure 2) as well as TCA cycle (supplementary Figure 3) - were analyzed more closely.

Table 1 shows that at least double as much genes of the KG1 cell line were down-regulated by both epigenetically active drugs and upregulated for all three sets of pathways.

Table 2 demonstrates that in the HL60 cell line up- and down-regulation were nearly balanced, only SAHA caused a 92 % increase in down-regulation ofthe TCA cycle.

The predominance of downregulated genes in the intra-mitochondrial pathways compared to glycolysis is shown in table 3.

Table 3 summarizes  the data from tables 1 and 2 in order to show the different influences of the drugs on down-regulation in the different cell lines and the predominance of downregulated genes in the intra-mitochondrial pathways as compared to cytoplasmic glycolysis.

However, it has to be mentioned, that none of these 3 pathways could be downregulated  to the level known for normal CD34⁺ cells (Figure 3A ), although treatment succeeded in single genes such as citrate synthase (CS) in KG1 cells (Figure 3B).

Figure 3A gives an overview of the overall expression and regulation of the leukemia cell lines before and after treatment. It becomes obvious, that all three pathways were up-regulated in the leukemia cell lines compared to the CD34+ cells and also that both epigenetically active drugs cause a down-regulation of all three pathways in both, the KG1 as well as the HL60 cell line.

Discussion

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Results of this study indicate that epigenetically active drugs induce a significant inhibition of cell multiplication in leukemia  cell lines. Interestingly, KG1 cells (Figure 2A and 2B) react more sensitive to drug treatment than HL60 cells (Figure 2C and 2D). Looking at figures 2A and 2C one notices that a 10-fold higher concentration  of DAC was needed for the same impact on a 50 % reduction of cell multiplication of the HL60 promyelocytic cell line as it had on the myeloblastic KG1 cell line (5 versus 0.5 µM). Thus, the HL60 exerted the same sensitivity to DAC as recently published for the HMC1 mastocytosis cell lines, independently  of presence of the D816V mutation of the KIT oncogene (Ghanim et al., 2012). This could indicate that the de-methylation of promoters from apoptosis-associated genes such as FAS and FASL (FAS-Ligand) is a critical mechanism in drug-mediated apoptosis-promotion, although additional pathways may also play a role (Lee et al., 2009; Soncini et al., 2012).

Focusing on the gene expression after treatment with the epigenetically active drugs DAC and SAHA, the results are quite intriguing: Many (but not all) metabolic relevant genes were downregulated. This fact suggests, on one hand, a return to normal cell metabolisms, even though the level of the healthy CD34+ cells was only rarely accomplished, and also an even more activated cell metabolism in those cell populations which survived the treatment, leading to cell cycle arrest and cell senescence.

Another outstanding finding is that DAC as well as SAHA generally cause a down-regulation of cell metabolism in the KG1 cell line and to a weaker extent in the HL60 cell line, where down-regulation is leveled by up-regulation, as about 50 % of the pathway relevant genes were up-regulated. These differences in gene expression after drug treatment indicate a specific activity of epigenetically active drugs on neoplastic stem- and progenitor cells. However, it has to be mentioned, that those cells, which survived treatment with demethylating drugs in HL60 cell line are already differentiated cells, which have lost their neoplastic potential and the weaker effect of the epigenetically active drugs on the HL60 cells may indicate a lower toxicity on more differentiated cell types.

The possibility that epigenetically active fusion-proteins, appearing as a consequence  of typical gene rearrangements in promyelocytic leukemia, interact with DNMT- or HDAC- inhibitors could be responsible for this observation (Uribesalgo et al., 2011) besides the possibility that differentiated cells exert a lower sensitivity to demethylating agents and that epigenetically active drugs have the potential to induce differentiation (Gozzini et al., 2005; Vlasakova et al., 2007).

Results of this study showed that the methylation inhibitor desoxy-azacytidine (DAC) and the histone-deacetylase inhibitor suberoyl anilide hydroxamic acid (SAHA) have different effects on the gene expression of the genes of the three main metabolic pathways, glycolysis, fatty b-oxidation and TCA cycle, in the more differentiated promyelocytic HL60 cell line than in the myeloblastic KG1 cell line. Our observation showing a lower rate of downregulation of glycolysis as compared to e.g. TCA cycle is confirmed by a recent publication (Caldero?n-Montan?o et al., 2013).

However, it has to be mentioned, that just a few genes (such as CS, Citrate Synthase, see also Figure 3B) could be down-regulated even to a lower level than in CD34+ cells by application of demethylating drugs. As citrate represents the basis for key metabolic pathways in neoplastic cells such as fatty acid synthesis (Schulze et al., 2012) (which is essential for cellular proliferation), this could provide an explanation for the anti-proliferative activity of epigenetic drugs as well as food components (Stefanska et al., 2012). In addition, the metabolic changes resulting from downregulation of CS could also promote the development of a phenotypic conversion als recently evidenced (Lin et al., 2012).

In conclusion,  data indicating that the KG1 cell line reacted more sensitive to demethylating drugs than the HL60 cell line, where drug treatment selected for survival of differentiated cells supports application of DAC and SAHA to leukemias with an undifferentiated phenotype but also with the potential to induce differentiation. Efficient targeting intra-mitochondrial pathways appears to be associated with multiple pro-apoptotic  activities of these drugs.

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Source(s) of Funding

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Acknowledgements: This study was supported by the Fonds zur Foerderung der Wissenschaftlichen Forschung (FWF; The Austrian Science Fund) Project P24370-B19, the Medical Scientific Fund of the Mayor of the City of Vienna (Project 2565), the WGKK (Social Health Insurance Vienna), and the AUVA (Austrian Social Insurance for Occupational Risks).

Competing Interests

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The other authors indicate that they do not have any potential conflicts of interest.

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