Energy Metabolism in H460 Lung Cancer Cells: Effects of Histone Deacetylase Inhibitors

et al. (2011) Energy Metabolism in H460 Lung Cancer Cells: Effects of Histone Deacetylase Inhibitors. PLoS ONE 6(7): e22264. doi:10.1371/journal.pone.0022264 Energy Metabolism in H460 Lung Cancer Cells: Effects of Histone Deacetylase Inhibitors Nvea Dias Amoe do 0 Mariana Figueiredo Rodrigues 0 Paula Pezzuto 0 Antonio Galina 0 Rodrigo Madeiro da 0 Costa 0 Fa bio Ceneviva Lacerda de Almeida 0 Tatiana El-Bacha 0 Franklin David Rumjanek 0 Alicia J. Kowaltowski, Instituto de Qumica - Universidade de Sao Paulo, Brazil 0 Instituto de Bioqu mica Me dica, Universidade Federal do Rio de Janeiro , Cidade Universita ria, Rio de Janeiro , Brazil Background: Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls. Methodology/Principal Findings: Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O2 consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged. Conclusion: NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype. - Funding: This work was supported by grants from Conselho Nacional de Desenvolvimento Cientfico e Tecnolo gico (CNPq), Fundacao de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundacao do Cancer, Brazilian Institute of Neuroscience - IBNnet FINEP, INCT - Excitotoxicity and Neuroprotection #01.06.0842 and INCT - Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The unchecked proliferation and invasion typical of cancer cells are processes that can only be sustained when there is sufficient energy supply, a feature that indicates the occurrence in transformed cells of distinct phenotypes that necessarily involve elements of the intermediary metabolism. In solid tumors it has been shown by Otto Warburg that cells have adapted to rely on anaerobic glycolysis as a strategy to maintain their prevailing anabolic status [1]. However, the upregulation of glycolysis exhibited by cancer cells does not necessarily imply a strict anaerobic phenotype nor a dysfunctional oxidative phosphorylation system (OXPHOS). Rather, it is believed that the normal interplay between the glycolysis in the cytosol and OXPHOS in the mitochondria becomes disturbed or reprogrammed in tumor cells. The Crabtree effect observed in cancer cells, or in rapidly proliferating cells exemplifies the intimate connection between glycolysis and the oxidative metabolism [2]. Interestingly, the anaerobic phenotype exhibited by cancer cells may in fact represent the cause rather than the consequence of the adaptive pressure. By considering that the glycolytic switch typical of cancer cells is acquired at the very onset of carcinogenesis, the idea arose that alterations in the glycolytic pathway may predispose cells to malignant transformation [3,4]. Selective advantages for the transformed cells could result from various features. For instance, it is known that hypoxia-inducible factor-1 (HIF-1a) greatly stimulates the expression of glucose and monocarboxylate transporters, glycolytic enzymes and induces a down regulation in pyruvate dehydrogenase complex [5]. Moreover, tumor cells present the isoform of HK that binds to the mitochondrial pore forming protein voltage-dependent anion channel (VDAC). By preventing the interaction of pro-apoptotic proteins with mitochondria the bound enzyme acts essentially as an anti-apoptotic agent. Indeed, it has been shown that the release of apoptotic proteins such as cytochrome c depends on the integrity of the Nterminal portion of VDAC [6]. Since it was demonstrated that HK and Bcl-2 were able to confer protection against apoptosis through interaction with the VDAC 1 N-terminal region, the participation of HK II as a promoter of cell differentiation was strengthened. Enzymes of the glycolytic and oxidative pathways are, as proteins in general, amenable to regulation of gene expression at the level of chromatin. Chromatin structures alternate between compacted and relaxed conformations which in turn depend on acetylation and deacetylation of the histone protein core. The enzymatic systems involved in these processes are histones acetyl transferases (HATs) that add acetyl groups to lysine residues and histone deacetylases (HDACs) that remove them. Compacted and relaxed chromatins have been linked to gene expression repression and activation, respectively. Although histones constitute the prime substrates for HATs and HDCAs, other non-histone proteins such as transcriptional factors-p53, pRb retinoblastoma protein and HIF-1a; chaperones (HSP90), metabolic enzymes (pyruvate kinase; acetyl-CoA synthase) and steroid receptors are also acetylated/deacetylated by these enzymes. Therefore, HATs and HDACs can affect a broad spectrum of biological processes that include growth arrest, DNA repair, cellular bioenergetics, cell death pathways (apoptosis, and autophagy), mitosis, generation of reactive oxygen species (ROS), senescence and angiogenesis[7,8]. Because of its repressive actions, HDACs have become interesting targets for the development of dr (...truncated)