Antonella Naldiniu,*, Irene Filippi', Elena Cinib, Manuela Rodriquez", Fabio Carrarou and Maurizio Taddeib "Department of Physiologt and bDepartment of Pharmaceutical and Applíed Chemistry, University of Siena, Via Aldo Moro, 53100 Sieia, haly; "bepirtmeni of Pharmaceutical Sciences, IJniversity of Salerno, Via Ponte Don Melillo, 84084 Fisciano (Salerno), Inly Abstract: The tumor microenvironment is characterized by a poor circulation which results in the selectionof neoplastic cells that can grow or survive under hypoxic conditions. The relationship betweenhypoxia and histone deacetylase (HDAC) inhibitors has been previously established. In ttris work we evaluatedthe effects of novel HDAC inhibitors (the natural peptide FR235222 and three Lnapeptiàe analogs) in the humanbreast cancer cell line MDAMB23l, culturedunder hypoxia (2o/o o.2= 14 mmHg) or normoxia (2oo/oO2x 140mmHg)' First, we found that the novel HDAC inhibiúors reduced cell proliferation in MDAMB23 I cells at an extentwhich was similar or even higher than that exerted by the classic HDAC inhibitors trichostatin-A and suberoylanilide hydroxarnicacid. More interestingly, the anìiproliferativeeffectsof the nqel HDAC inhibitors were, in general, significantly higher in hyporriccells than in normoxic controls. ' Hypoxic MDAMB23I cells expressed high levelsof the hypoxia-inducible factor(HIF)-Ia and HlF-la-related genes, suchas vascular endothelial grourth facúor,Bcl-2/ElB lSkDa interacting protein-3, glucose transporter-I, carbonic anhydrase IX, as determinedby Westem blof analysisand qRT-PCR. Finally, we found that HIF-lc and HlF-lu-related genes were significantly downregulated by FR235222and anilogs. h òonclusion, the identification of novel effects exertedby the HDAC inhibitors, characterized by a strong efficacy in inhibiting-the expression of HIF-la and its relatedgenes, may have importantimplicationsin the pharmacological control of several tumors, including breast cancer, characterized by the presence of hypoxia,angiogenesis and metabolic derangements. Keywords: Angiogenesis, Anticancer drugs, Breast cancer, Carbonic anhydrase,Cell proliferation, Epigenetic regulation, Gene expression, Glucose transportor, Hypoxia, Hypoxia-inducible factor (HIF)-lalpha, Histone deacetylase inhibitors, Metabolisrry Transcription factors, Tumor micro environment, Vascular endothelial growth facto r. Anti-Cancer Agents in Medicínal Chemístry, 2012, 12, 407413 Downregulation of Hypoxia-related Responses by Novel Deacetylase Inhibitors in MDAMB}3I Breast Cancer Cells 407 Antitumor Histone (HDAC) inhibitors downregulaùe HIF-Ia and exert potent antiangiogenic effects [3,14]. HDAC inhibitors affect cell cycle progression [5,16] and inducecell differentiation, apoptosis and inhibition of tumor growth in vivo [17]. Although the mechanism involved in HDAC-mediated HIF downregulationhas not been fully elucidated [8], several molecules have been identified as epigenetic modulators of HIF (Fig. 1). Most of them belongto the hydroxamic acid class, which includes Trichostatin'A (TSA) or suberoylanilide hydroxamic acid (SAHA, alreadyregistered as an anticancer drug Vorinostat). Fig. (l) also includes FK288, registered as Romidepsin and active after in viw cleavage of the disulphide bond, andthe amide MS-275 [19,20]. Recently, several tetrapeptides structurally related to natural peptide FR235222 have beenidentifred as a new classof HDAC inhibitors [21]. Studies conductedin human leukemia cell lines revealed that FR235222 inhibits cell proliferation to an extent similar to the more toxic TSA [22]. To establishwhether these novel HDAC inhibitors, characterized by promising physicochemical properties and plasma concenration levels [23], were able to modulate HIF-I expression, hypoxia-related responses, as well as cell proliferation, we decided to study the effects of the natural FR235222 and threeanalogs (Al, A2 and A3) in the breast cancer cell line MDAMB23l.In addition, the hypoxiccondition of tumor cells is one of the main stimulators of angiogenesis in many solid tumors [24] and reportson the biological effects exertedby HDAC inhibitors in hypoxic breastcancercells are scant.ln this work we show that MDAMB23I cell proliferation was inhibited by the novel HDAC inhibitors FR235222 and analogs to an extent which was similar or EVen higher than that inducedby T$A and SAHA. More interestingly, hypoxia potentiates the antiproffierative effecrsof HDAC inhibitors in MDAMB23I. Finally, we show that FR235222 and analogs downregulateHIF-1c accumulation and HlF-l-responsive gene expression in MDAMB23I breast cancer cells. @2012 Bentham Science Publishers INTRODUCTION The tumor microenvironment is characterizedby a poor circulationwhich results in the selection of neoplastic cells that can gfow or survive under hypoxic conditions [l]. The response to a decreased oxygen tension, occurringalso in inflamed tissues and in normal development and physiology [2], results in adaptationally appropriate alterations of geneexpression throughthe ranscription factor hypoxia-inducible factor (HIF)-I ùo overcome any shortage of oxygen. These adaptations include the activation of HIF-I- dependent genes that control angiogenesis and facilitate anaerobic glycolysis [3-5]. Thus, drugs that inhibit HIF-I expression have grcat potentialasantitumor agents. HIF-I is an crp-heterodimer and both the HIF-a and HIF-p subunitsexist as a seriesof isoforms encoded by distinct genetic loci [3-5]. HIF-IB can dimerize with different basic helix-loop- helix-PAS proteins and is constitutive,while HIF-a subunitsare inducible by hypoxia.Among threeHIF-a isoforms, HIF-lo and HIF-2c appearto be closely related and are able to interactwith hypoxiaresponse elements (HREs)to induce transcriptional activity [6]. However, in solid tumors, such as in breast cancer,HIF-1c represents the primary source of hypoxic transcriptional responses [7,8]. The regulation of HIF is complex and involves changes at transcriptional, posttranscriptional and posttranslational levels in response to hypoxia. Hydroxylation, ubiquitination,S-nitrosation, phosphorylation and acetylation have been shown to determineits halfJife and/ortranscriptional activity of HIF-Ic [9]. The role of acetylation in controlling HIF-la functionhasbeenthe objective of intensive investigation [l0-12], suggesting that histone deacetylase *Address correspondence to this author at the Department of PhysiÓlogy' University of Siena,Via Aldo Moro, 53100 SIENA, ltaly; Tel: 0039-0577 -234212: Fax:0039-05'7 7 -234219; E-mail: Naldini@Unisi. it 187 15992 I 12 $58.00+.00