An assessment of crucial structural contributors of HDAC6 inhibitors through fragment-based non-linear pattern recognition and molecular dynamics simulation approaches.

Amidst the Zn2+-dependant isoforms of the HDAC family, HDAC6 has emerged as a potential target associated with an array of diseases, especially cancer and neuronal disorders like Rett's Syndrome, Alzheimer's disease, Huntington's disease, etc. Also, despite the availability of a handful of HDAC inhibitors in the market, their non-selective nature has restricted their use in different disease conditions. In this situation, the development of selective and potent HDAC6 inhibitors will provide efficacious therapeutic agents to treat different diseases. In this context, this study has been carried out to evaluate the potential structural contributors of quinazoline-cap-containing HDAC6 inhibitors via machine learning (ML), conventional classification-dependant QSAR, and MD simulation-based binding mode of interaction analysis toward HDAC6 binding. This combined conventional and modern molecular modeling study has revealed the significance of the quinazoline moiety, substitutions present at the quinazoline cap group, as well as the importance of molecular property, number of hydrogen bond donor-acceptor functions, carbon-chlorine distance that significantly affects the HDAC6 binding of these inhibitors, subsequently affecting their potency . Interestingly, the study also revealed that the substitutions such as the chloroethyl group, and bulky quinazolinyl cap group can affect the binding of the cap function with the amino acid residues present in the loops proximal to the catalytic site of HDAC6. Such contributions of cap groups can lead to both stabilization and destabilization of the cap function after occupying the hydrophobic catalytic site by the aryl hydroxamate linker-ZBG functions.

Assessment of suberoylanilide hydroxamic acid on a Alzheimer's disease model induced by ß-amyloid(1-42) in aged female mice: Neuromodulatory and epigenetic effect.

Alzheimer's disease (AD) is a neurodegenerative disease that affects several elderly people per years. AD is a pathology of multifactorial etiology, resulting from multiple environmental and genetic determinants. However, there is no effective pharmacological alternative for the treatment of this illness. In this sense, the purpose of current study was to characterize the mechanisms by which Aß1-42 injection via intracerebroventricular induces neurobehavioral changes in a time-course curve. In addition, suberoylanilide hydroxamic acid (SAHA) inhibitor of histone deacetylase (HDAC) was used to investigate the involvement of epigenetic modifications Aß1-42-caused in aged female mice. In general manner, Aß1-42 injection induced a major neurochemical disturbance in hippocampus and prefrontal cortex of animals and a serious impairment of memory. Overall, SAHA treatment attenuated neurobehavioral changes caused by Aß1-42 injection in aged female mice. The subchronic effects presented of SAHA were through modulation of HDAC activity, regulation of brain-derived neurotrophic factor (BDNF) levels and expression of BDNF mRNA, accompanied by unlocking cAMP/PKA/pCREB pathway in hippocampus and prefrontal cortex of animals.

Assessment of HDAC Inhibitor-Induced Endoplasmic Reticulum (ER) Stress.

The endoplasmic reticulum (ER) is a multifunctional cell organelle which is important for the folding and processing of proteins. Different endogenous and exogenous factors can disturb the ER homeostasis, causing ER stress and activating the unfolded protein response (UPR) to remove misfolded proteins and aggregates. ER stress and the UPR are associated with several human diseases, such as diabetes, Alzheimer's or Parkinson's disease, and cancer. Histone deacetylase inhibitors (HDACi) are used to treat cancer and were shown to induce ER stress/to modulate the UPR, although the exact mechanism is not fully understood and needs further research. Several approaches to monitoring ER stress exist. Here we describe methods including qPCR, Western blot, transmission electron microscopy, and fluorescence microscopy to analyze changes in mRNA and protein expression levels as well as defects in ER structures after HDAC inhibitor-induced ER stress.

Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition.

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.

A comparative quantitative structural assessment of benzothiazine-derived HDAC8 inhibitors by predictive ligand-based drug designing approaches.

Histone deacetylase 8 (HDAC8) is a verified biomolecular target associated with diverse diseases including cancer. Though several HDAC inhibitors emerged effective against such diseases, no selective HDAC8 inhibitor is approved to date. Therefore, the development of potent HDAC8-selective inhibitors is inevitable to combat such diseases. Here, some benzothiazine-derived HDAC8 inhibitors were considered for a comparative QSAR analysis which may elucidate the prime structural components responsible for modulating their efficacy. Several outcomes from these diverse modelling techniques justified one another and thus validated each other. The ligand-based pharmacophore modelling study identified ring aromatic, positive ionizable, and hydrophobic features as essential structural attributes for HDAC8 inhibition. Besides, MLR, HQSAR and field-based 3D-QSAR studies signified the utility of the positive ionizable and hydrophobic features for potent HDAC8 inhibition. Again, the field-based 3D-QSAR study provided useful insight regarding the substitution in the fused phenyl ring. Moreover, the current observations also validated the previously reported molecular docking observations. Based on the outcomes, some new molecules were designed and predicted. Therefore, this comparative structural analysis of these HDAC8 inhibitors will surely assist in the development of potent HDAC8 inhibitors as promising anticancer therapeutics in the future.

Assessment of Pharmacological Interactions between SIRT2 Inhibitor AGK2 and Paclitaxel in Different Molecular Subtypes of Breast Cancer Cells.

Breast carcinoma (BC) is the most commonly diagnosed type of cancer in women in the world. Although the advances in the treatment of BC patients are significant, numerous side effects, severe toxicity towards normal cells as well as the multidrug resistance (MDR) phenomenon restrict the effectiveness of the therapies used. Therefore, new active compounds which decrease the MDR, extend disease-free survival, thereby ameliorating the effectiveness of the current treatment regimens, are greatly needed. Histone deacetylase inhibitors (HDIs), including sirtuin inhibitors (SIRTi), are the epigenetic antitumor agents which induce a cytotoxic effect in different types of cancer cells, including BC cells. Currently, combined forms of therapy with two or even more chemotherapeutics are promising antineoplastic tools to obtain a better response to therapy and limit adverse effects. Thus, on the one hand, much more effective chemotherapeutics, e.g., sirtuin inhibitors (SIRTi), are in demand; on the other hand, combinations of accepted cytostatics are trialed. Thus, the aim of our research was to examine the combination effects of a renowned cytotoxic drug paclitaxel (PAX) and SIRT2 inhibitor AGK2 on the proliferation and viability of the T47D, MCF7, MDA-MB-231, MDA-MB-468, BT-549 and HCC1937 BC cells. Moreover, cell cycle arrest and apoptosis induction were explored. The type of pharmacological interactions between AGK2 and PAX in different molecular subtypes of BC cells was assessed using the advanced isobolographic method. Our findings demonstrated that the tested active agents singly inhibited viability and proliferation of BC cells as well as induced cell cycle arrest and apoptosis in the cell-dependent context. Additionally, AGK2 increased the antitumor effect of PAX in most BC cell lines. We observed that, depending on the BC cell lines, the combinations of tested drugs showed synergistic, additive or antagonistic pharmacological interaction. In conclusion, our studies demonstrated that the consolidated therapy with the use of AGK2 and PAX can be considered as a potential therapeutic regimen in the personalized cure of BC patients in the future.

Cracking a cancer code histone deacetylation in epigenetic: the implication from molecular dynamics simulations on efficacy assessment of histone deacetylase inhibitors.

Epigenetic changes, histone acetylation and deacetylation in chromatin have been intensively studied due to their significance in regulating the gene expression. According to the type of tumor, the levels of histone deacetylases (HDAC) are varied. HDAC inhibitors are a new promising class of compounds that inhibit the proliferation of tumor cells. In this study, the inhibitory efficacy of some HDAC inhibitors such as vorinostat, panobinostat, abexinostat, belinostat, resminostat, dacinostat and pracinostat was studied using molecular dynamics simulation. The inhibitory efficacy was estimated by computing the enzyme's stability, positional stability of the individual amino acids and interaction energies of HDLP-inhibitor complexes. It is hoped that this investigation may improve our understanding of the atomic-level description of the inhibitor binding site and how the HDAC inhibitors change the environment of the enzyme's active site. The results obtained from the root-mean-square deviation, the radius of gyration, solvent-accessible surface area, root-mean-square fluctuation, stride server and Ramachandran plot have revealed that the stability of HDLP enzyme with vorinostat, panobinostat and abexinostat is higher than the other studied complexes. According to the calculated values for MM-PBSA, LIE, semi-LIE binding free energies and interaction energies, the stability of the HDLP enzyme varies as panobinostat > abexinostat > vorinostat where resminostat complex showed relatively low stability. The ligandability and drugability values also give the same trend as above. The findings revealed that the panobinostat and abexinostat are potential lead compounds as reference inhibitor vorinostat. Therefore, it is possible to use these drugs as HDAC inhibitors in clinical practices. Also, the outcomes of this study could be utilized to identify new inhibitors for clinical research.Communicated by Ramaswamy H. Sarma.

Development of decision trees to discriminate HDAC8 inhibitors and non-inhibitors using recursive partitioning.

Histone deacetylase 8 (HDAC8) is involved in malignancy. Overexpression of HDAC8 is correlated with various cancers. Design of selective HDAC8 inhibitors is always a challenging task to the chemistry audiences. In this communication, a diverse set comprising large number of compounds are subjected to recursive partitioning (RP) analysis to develop decision trees to discriminate compounds into HDAC8 inhibitors (active) and non-inhibitors (inactive). Acquiring knowledge about the essential structural and physicochemical parameters can be useful in designing potential and selective HDAC8 inhibitors. Moreover, this work validates our previous results observed in Bayesian modelling study of this dataset. This comparative learning will surely enrich drug discovery aspects related to HDAC8 inhibitors.Communicated by Ramaswamy H. Sarma.

Integrated Assessment of Viral Transcription, Antigen Presentation, and CD8+ T Cell Function Reveals Multiple Limitations of Class I-Selective Histone Deacetylase Inhibitors during HIV-1 Latency Reversal.

Clinical trials investigating histone deacetylase inhibitors (HDACi) to reverse HIV-1 latency aim to expose reservoirs in antiretroviral (ARV)-treated individuals to clearance by immune effectors, yet have not driven measurable reductions in the frequencies of infected cells. We therefore investigated the effects of the class I-selective HDACi nanatinostat and romidepsin on various blocks to latency reversal and elimination, including viral splicing, antigen presentation, and CD8+ T cell function. In ex vivo CD4+ T cells from ARV-suppressed individuals, both HDACi significantly induced viral transcription, but not splicing nor supernatant HIV-1 RNA. In an HIV-1 latency model using autologous CD8+ T cell clones as biosensors of antigen presentation, neither HDACi-treated CD4+ T cell condition induced clone degranulation. Both HDACi also impaired the function of primary CD8+ T cells in viral inhibition assays, with nanatinostat causing less impairment. These findings suggest that spliced or cell-free HIV-1 RNAs are more indicative of antigen expression than unspliced HIV-RNAs and may help to explain the limited abilities of HDACi to generate CD8+ T cell targets in vivoIMPORTANCE Antiretroviral (ARV) drug regimens suppress HIV-1 replication but are unable to cure infection. This leaves people living with HIV-1 burdened by a lifelong commitment to expensive daily medication. Furthermore, it has become clear that ARV therapy does not fully restore health, leaving individuals at elevated risk for cardiovascular disease, certain types of cancers, and neurocognitive disorders, as well as leaving them exposed to stigma. Efforts are therefore under way to develop therapies capable of curing infection. A key focus of these efforts has been on a class of drugs called histone deacetylase inhibitors (HDACi), which have the potential of exposing hidden reservoirs of HIV-1 to elimination by the immune system. Unfortunately, clinical trial results with HDACi have thus far been disappointing. In the current study, we integrate a number of experimental approaches to build a model that provides insights into the limited activity of HDACi in clinical trials and offers direction for future approaches.

First example of Azo-Sulfa conjugated chromene moieties: Synthesis, characterization, antimicrobial assessment, docking simulation as potent class I histone deacetylase inhibitors and antitumor agents.

This report presents the development of a novel and primary model of sulfonamide compounds encompassing a chromene azo motif with the intent of becoming applicable for drug candidates in the cases of drug-resistant pathogens. The novel molecules (7a-n) have been synthesized via a two-step reaction. First, 4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e) were obtained through the reaction of their corresponding diazotized 4-aminobenzenesulfonamides (1a-e) with resorcinol, followed by the heterocyclization of 3a-e with arylidenemalononitriles (6a-d). Upon structural identification, the newly synthesized compounds were evaluated for their antibacterial and antifungal activities. Moreover, their cytotoxic screening was performed against three cancer cell lines: HCT-116, HepG-2, and MCF-7. Further examinations were comprised of the inhibitory effect analyses of the novel sulfonamide/chromene derivatives against the HDAC classes and the Tubulin polymerization in order to discern the prime antitumor drug candidates.

Assessment of SIRT2 Inhibitors in Mouse Models of Cancer.

New therapeutics directed against established and novel molecular targets are urgently needed to intervene against cancer. Recently, it was reported that several members of the sirtuin family (SIRT1-7), the mammalian orthologs of the silent information regulator 2 (Sir2) protein in Saccharomyces cerevisiae, play important roles in carcinogenesis. Although SIRT2 has been attributed both tumor-promoting and tumor-suppressing activities in different contexts, selective SIRT2 inhibition with a small molecule mechanism-based inhibitor known as Thiomyristoyl lysine (TM) repressed the growth of breast cancer cell lines. In light of the anticancer effect of SIRT2 inhibition in cell culture, it was critical to assess the efficacy of TM as a potential anticancer therapy in vivo. This was accomplished by testing the SIRT2 inhibitor in genetically engineered and xenotransplantation mouse models of breast cancer, using the procedures detailed in this chapter.

Preclinical assessment of histone deacetylase inhibitor quisinostat as a therapeutic agent against esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most serious life-threatening malignancies. Although chemotherapeutic targets and agents for ESCC have made much progress recently, the efficacy is still unsatisfactory. Therefore, there is still an unmet medical need for patients with ESCC. Here, we report the expression status of HDAC1 in human ESCC and matched paracancerous tissues, and the results indicated that HDAC1 was generally upregulated in ESCC specimens. Furthermore, we comprehensively assessed the anti-ESCC activity of a highly active HDAC1 inhibitor quisinostat. Quisinostat could effectively suppress cellular viability and proliferation of ESCC cells, as well as induce cell cycle arrest and apoptosis even at low treatment concentrations. The effectiveness was also observed in KYSE150 xenograft model when quisinostat was administered at tolerated doses (3 mg/kg and 10 mg/kg). Meanwhile, quisinostat also had the ability to suppress the migration and invasion (pivotal steps of tumor metastasis) of ESCC cells. Western blot analysis indicated that quisinostat exerted its anti-ESCC effects mainly through blockade of Akt/mTOR and MAPK/ERK signaling cascades. Overall, HDAC1 may serve as a potential therapeutic target for ESCC, and quisinostat deserves to be further assessed as a promising drug candidate for the treatment of ESCC.

Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine. Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PD-L1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi. Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response.

Assessment of HDACi-Induced Protein Cleavage by Caspases.

Aberrant histone deacetylase (HDAC) activity often correlates with neoplastic transformation and inhibition of HDACs by small molecules has emerged as a promising strategy to treat hematological malignancies in particular. Treatment with HDAC inhibitors (HDACis) often prompts tumor cells to undergo apoptosis, thereby causing a caspase-dependent cleavage of target proteins. An unexpectedly large number of proteins are in vivo caspase substrates and defining caspase-mediated substrate specificity is a major challenge. In this chapter we demonstrate that the hematopoietic transcription factor PU.1 becomes cleaved after treatment of acute myeloid leukemia (AML) cells with the HDACis LBH589 (panobinostat) or MS-275 (entinostat). To define caspase specificity for PU.1, an in vitro caspase assay including caspases 1-10 with in vitro-translated PU.1 is described in detail.

Assessment of HDACi-Induced Cytotoxicity.

The chromatin contains the genetic and the epigenetic information of a eukaryotic organism. Posttranslational modifications of histones, such as acetylation and methylation, regulate their structure and control gene expression. Histone acetyltransferases (HATs) acetylate lysine residues in histones while histone deacetylases (HDACs) remove this modification. HDAC inhibitors (HDACi) can alter gene expression patterns and induce cytotoxicity in cancer cells. Here we provide an overview of methods to determine the cytotoxic effects of HDACi treatment. Our chapter describes colorimetric methods, like trypan blue exclusion test, crystal violet staining, lactate dehydrogenase assay, MTT and Alamar Blue assays, as well as fluorogenic methods like TUNEL staining and the caspase-3/7 activity assay. Moreover, we summarize flow cytometric analysis of propidium iodide uptake, annexin V staining, cell cycle status, ROS levels, and mitochondrial membrane potential as well as detection of apoptosis by Western blot.

Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry.

Posttranslational acetylation of lysine residues has been discovered as multifaceted regulatory modification for various nuclear, cytoplasmic, and mitochondrial proteins. The implementation of high-resolution and high-throughput mass spectrometry (MS) approaches has led to the identification of a hitherto underappreciated, large number of acetylation sites for a broad spectrum of cellular proteins. In this chapter, we describe a comprehensive protocol for the purification of an in vivo-acetylated, ectopically expressed, FLAG-epitope tagged nonhistone protein through immunoprecipitation (IP). The protocol also covers the sample preparation by SDS-PAGE, proteolytic digestion, and the analysis by LC-ESI MS. The success of this methodology, however, strongly depends on the physico-chemical properties of the respective protein(s) and the quality of selected peptide mass spectra.

Generation and Assessment of Fusions Between HDACi and TKi.

Chimeric compounds combine the structural features of inhibitors of histone deacetylases (HDACi) and tyrosine kinase inhibitors (TKi), and therefore unite the effects of a dual-targeting strategy in one compound. Here, we describe the generation of such hybrid molecules. Small molecules, known as TKi, are combined with a Zn2+ chelating motive, preferentially a hydroxamic acid, in addition. The resulting small molecules also can inhibit histone deacetylases, which are dependent on the catalytically active Zn2+. Moreover, we summarize how the growth-inhibitory effects of these combined compounds can be determined with a simple proliferation assay with a leukemic cell line.

Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors.

The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6 inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit α-tubulin deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the pursuit of novel potent and selective HDAC6 inhibitors.

An Automated Strategy for Binding-Pose Selection and Docking Assessment in Structure-Based Drug Design.

Molecular docking is a widely used technique in drug design to predict the binding pose of a candidate compound in a defined therapeutic target. Numerous docking protocols are available, each characterized by different search methods and scoring functions, thus providing variable predictive capability on a same ligand-protein system. To validate a docking protocol, it is necessary to determine a priori the ability to reproduce the experimental binding pose (i.e., by determining the docking accuracy (DA)) in order to select the most appropriate docking procedure and thus estimate the rate of success in docking novel compounds. As common docking programs use generally different root-mean-square deviation (RMSD) formulas, scoring functions, and format results, it is both difficult and time-consuming to consistently determine and compare their predictive capabilities in order to identify the best protocol to use for the target of interest and to extrapolate the binding poses (i.e., best-docked (BD), best-cluster (BC), and best-fit (BF) poses) when applying a given docking program over thousands/millions of molecules during virtual screening. To reduce this difficulty, two new procedures called Clusterizer and DockAccessor have been developed and implemented for use with some common and "free-for-academics" programs such as AutoDock4, AutoDock4(Zn), AutoDock Vina, DOCK, MpSDockZn, PLANTS, and Surflex-Dock to automatically extrapolate BD, BC, and BF poses as well as to perform consistent cluster and DA analyses. Clusterizer and DockAccessor (code available over the Internet) represent two novel tools to collect computationally determined poses and detect the most predictive docking approach. Herein an application to human lysine deacetylase (hKDAC) inhibitors is illustrated.

Assessment of Interactions between Cisplatin and Two Histone Deacetylase Inhibitors in MCF7, T47D and MDA-MB-231 Human Breast Cancer Cell Lines - An Isobolographic Analysis.

Histone deacetylase inhibitors (HDIs) are promising anticancer drugs, which inhibit proliferation of a wide variety of cancer cells including breast carcinoma cells. In the present study, we investigated the influence of valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), alone or in combination with cisplatin (CDDP) on proliferation, induction of apoptosis and cell cycle progression in MCF7, T47D and MDA-MB-231 human breast carcinoma cell lines. The type of interaction between HDIs and CDDP was determined by an isobolographic analysis. The isobolographic analysis is a very precise and rigorous pharmacodynamic method, to determine the presence of synergism, addition or antagonism between different drugs with using variety of fixed dose ratios. Our experiments show that the combinations of CDDP with SAHA or VPA at a fixed-ratio of 1:1 exerted additive interaction in the viability of MCF7 cells, while in T47D cells there was a tendency to synergy. In contrast, sub-additive (antagonistic) interaction was observed for the combination of CDDP with VPA in MDA-MB-231 "triple-negative" (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) human breast cancer cells, whereas combination of CDDP with SAHA in the same MDA-MB-231 cell line yielded additive interaction. Additionally, combined HDIs/CDDP treatment resulted in increase in apoptosis and cell cycle arrest in all tested breast cancer cell lines in comparison with a single therapy. In conclusion, the additive interaction of CDDP with SAHA or VPA suggests that HDIs could be combined with CDDP in order to optimize treatment regimen in some human breast cancers.