Dual targeting of epigenetic therapy in cancer

Aberrant epigenetic silencing of tumor suppressor genes by promoter DNA hypermethylation and histone deacetylation plays an important role in the pathogenesis of cancer. The potential reversibility of epigenetic abnormalities encouraged the development of pharmacologic inhibitors of DNA methylation and histone deacetylation as anti-cancer therapeutics. (Pre)clinical studies of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have yielded encouraging results, especially against hematologic malignancies. Recently, several studies demonstrated that DNMT and HDAC inhibitors are also potent angiostatic agents, inhibiting (tumor) endothelial cells and angiogenesis in vitro and in vivo. By reactivation of epigenetically silenced tumor suppressor genes with angiogenesis inhibiting properties, DNMT and HDAC inhibitors might indirectly - via their effects on tumor cells - decrease tumor angiogenesis in vivo. However, this does not explain the direct angiostatic effects of these agents, which can be unraveled by gene expression studies and examination of epigenetic promoter modifications in endothelial cells treated with DNMT and HDAC inhibitors. Clearly, the dual targeting of epigenetic therapy on both tumor cells and tumor vasculature makes them attractive combinatorial anti-tumor therapeutics. Here we review the therapeutic potential of DNMT and HDAC inhibitors as anti-cancer drugs, as evaluated in clinical trials, and their angiostatic activities, apart from their inhibitory effects on tumor cells.     

Clinical Potential of Histone Deacetylase Inhibitors as Stand Alone Therapeutics and in Combination with other Chemotherapeutics or Radiotherapy for Cancer

Histone deacetylase inhibitors are emerging as a new class of cancer chemotherapeutics and already are being heralded as the first anti-cancer drugs targeting the epigenome. Through histone hyperacetylation-mediated changes in chromatin conformation and gene expression, histone deacetylase inhibitors induce differentiation, cell cycle arrest, apoptosis, growth inhibition and cell death, which are more pronounced in transformed cell-lines than in normal cells. Additional anti-cancer effects of HDAC inhibitors include inhibition of migration, invasion and angiogenesis in vivo. Indeed, clinical anti-cancer activity has been observed using HDAC inhibitors as single agents or in combination with conventional chemotherapeutics, in phase I and II trials. Furthermore, numerous pre-clinical studies are suggesting a potential clinical role for HDAC inhibitors in radiotherapy either as radiation sensitizers or protectors. In this article the molecular basis for the clinical potential of HDAC inhibitors, either as stand alone cancer therapeutics or in combination with other chemotherapy agents or ionizing radiation will be overviewed.      

Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors

Histone deacetylases (HDACs) are involved in post-translational modification and gene expression. Cancer cells recruited amounts of HDACs for their survival by epi-genetic down regulation of tumor suppressor genes. HDACs have been the promising targets for treatment of cancer, and many HDAC inhibitors have been investigated nowadays. In previous study, we synthesized δ-lactam core HDAC inhibitors which showed potent HDAC inhibitory activities as well as cancer cell growth inhibitory activities. Through QSAR study of the δ-lactam based inhibitors, the smaller core is suggested as more active than larger one because it fits better in narrow hydrophobic tunnel of the active pocket of HDAC enzyme. The smaller γ-lactam core HDAC inhibitors were designed and synthesized for biological and property optimization. Phenyl, naphthyl and thiophenyl groups were introduced as the cap groups. Hydrophobic and bulky cap groups increase potency of HDAC inhibition because of hydrophobic interaction between HDAC and inhibitors. In overall, γ-lactam based HDAC inhibitors showed more potent than δ-lactam analogues.     

HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation

Therapeutic effects of histone deacetylase (HDAC) inhibitors in cancer models were first linked to their ability to cause growth arrest and apoptosis of tumor cells. It is now clear that these agents also have pleiotropic effects on angiogenesis and the immune system, and some of these properties are likely to contribute to their anti-cancer activities. It is also emerging that inhibitors of specific HDACs affect the differentiation, survival and/or proliferation of distinct immune cell populations. This is true for innate immune cells such as macrophages, as well as cells of the acquired immune system, for example, T-regulatory cells. These effects may contribute to therapeutic profiles in some autoimmune and chronic inflammatory disease models. Here, we review our current understanding of how classical HDACs (HDACs 1-11) and their inhibitors impact on differentiation, survival and proliferation of distinct leukocyte populations, as well as the likely relevance of these effects to autoimmune and inflammatory disease processes. The ability of HDAC inhibitors to modulate leukocyte survival may have implications for the rationale of developing selective inhibitors as anti-inflammatory drugs.     

Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors

Histone deacetylases (HDACs) have proven to be promising targets for the development of anti-cancer drugs. In this study, we reported a series of novel chalcone based tubulin and HDAC dual-targeting inhibitors. Three compounds inhibited the activities of HDAC and tubulin polymerization simultaneously and displayed anti-proliferative activities toward eleven human tumor cell lines. Compound 8a remarkably induced growth inhibition, apoptosis and G2/M phase arrest of A549 tumor cells. Finally, the inhibitory activities of 8a against HDAC6 and tubulin were rationalized by molecular docking studies.     

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC₅₀ values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.     

Anticancer Activity of MPT0E028, a Novel Potent Histone Deacetylase Inhibitor, in Human Colorectal Cancer HCT116 Cells In Vitro and In Vivo

Recently, histone deacetylase (HDAC) inhibitors have emerged as a promising class of drugs for treatment of cancers, especially subcutaneous T-cell lymphoma. In this study, we demonstrated that MPT0E028, a novel N-hydroxyacrylamide-derived HDAC inhibitor, inhibited human colorectal cancer HCT116 cell growth in vitro and in vivo. The results of NCI-60 screening showed that MPT0E028 inhibited proliferation in both solid and hematological tumor cell lines at micromolar concentrations, and was especially potent in HCT116 cells. MPT0E028 had a stronger apoptotic activity and inhibited HDACs activity more potently than SAHA, the first therapeutic HDAC inhibitor proved by FDA. In vivo murine model, the growth of HCT116 tumor xenograft was delayed and inhibited after treatment with MPT0E028 in a dose-dependent manner. Based on in vivo study, MPT0E028 showed stronger anti-cancer efficacy than SAHA. No significant body weight difference or other adverse effects were observed in both MPT0E028-and SAHA-treated groups. Taken together, our results demonstrate that MPT0E028 has several properties and is potential as a promising anti-cancer therapeutic drug.     

p21waf1/Cip1 partially mediates apoptosis in hepatocellular carcinoma cells

p21waf1/Cip1 (p21) is a tumor suppressor gene involved in apoptosis in many cancer cell types induced by different agents. In spite of concomitant induction of p21 by many anti-cancer drugs, including inhibitors of histone deacetylases (HDACi), its pro-apoptotic action has been debated during the last several years due to a lack of direct evidence regarding the exact role of p21 in apoptosis. With the help of anti-sense p21 expression, we show here that p21 is mediating the apoptotic effects of HDACi 4-phenylbutyrate (4-PB) and Trichostatin A (TSA) on the hepatocellular hepatocarcinoma Hep3B cells. Hep3B cells were transfected by EGFP-p21 anti-sense or sense plasmids, and apoptosis induced by HDACi was assessed by TUNEL assay. The results show that the p21 anti-sense construct prevents apoptosis, induced by HDAC inhibitors in Hep3B cells. The obtained results suggest an important role for p21 in mediating the apoptotic effect of HDACi.     

MS-275, a potent orally available inhibitor of histone deacetylases--the development of an anticancer agent

In the last few years it was found that beside genetic aberrations, epigenetic changes also play an important role in tumorigenesis. Acetylation and deacetylation of histones have been found to contribute to a significant extent to epigenetic regulation of gene expression. Analyses of various tumor models and patient samples revealed that the enzyme class of histone deacetylases is associated with many types of cancer and that, for example, over-expression of these enzymes leads to a disturbed balance between acetylation and deacetylation of histones, resulting in differences in the gene expression patterns between normal and cancer cells. Consequently, this class of enzymes has been considered as a potential target for cancer therapy. Numerous inhibitors have been identified and several are in clinical development. Although, with SAHA, one inhibitor has been approved by the FDA for a tumor indication, many open questions remain regarding the mode of action of these inhibitors. In this review, various aspects of preclinical and clinical research of the HDAC inhibitor MS-275 are described, to provide insight into the development of such a compound.     

Histone deacetylases: a new class of efficient anti-tumor drugs

Circa twenty-five years ago, cancer research was dominated by the concept that the origin of cancer was genetic. Thousands of genetic alterations have indeed been identified involving more than hundred different genes in cancer development. Today, the model has evolved: it has been demonstrated that malignancies can be initiated not only through genetic alterations but also through epigenetic deregulations. By altering the expression of gene involved in cell regulation, epigenetic alterations, such as histone acetylation, play a key role in the initiation and progression of neoplasm. It has been shown that an imbalance between the acelylated and deacetylated status of chromatin is significantly involved in the acquisition of a malignant phenotype. Thus, the modulation of the histone acetylation level by histone deacetylase (HDAC) inhibitors could lead to a genetic re-programmation in cancer cells that would favor apoptosis and prevent proliferation. The potential therapeutic value of several HDAC inhibitors for cancer patients has been evaluated in clinical assays with very promising outcome. Indeed, the first inhibitors available for patients has been recently approved for cancer patients tracing the way for a new class of promising anti-cancer therapy modalities.     

Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTX’s inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.     

Inhibitors of histone deacetylase as antitumor agents: A critical review

Histone deacetylase (EC 3.5.1.98 – HDAC) is an amidohydrolase involved in deacetylating the histone lysine residues for chromatin remodeling and thus plays a vital role in the epigenetic regulation of gene expression. Due to its aberrant activity and over expression in several forms of cancer, HDAC is considered as a potential anticancer drug target. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as cell survival, differentiation and apoptosis in tumor cells and thus exhibit anticancer activity. Till date, four drugs, namely Vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101) and Panobinostat (LBH-589) have been granted FDA approval for cancer and several HDAC inhibitors are currently in various phases of clinical trials, either as monotherapy and/or in combination with existing/novel anticancer agents. Regardless of this, today scientific efforts have fortified the quest for newer and novel HDAC inhibitors that show isoform selectivity. This review focuses on the chemistry of the molecules of two classes of HDAC inhibitors, namely short chain fatty acids and hydroxamic acids, investigated so far as novel therapeutic agents for cancer.     

A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.     

Dose-dependent blockade to cardiomyocyte hypertrophy by histone deacetylase inhibitors

Postnatal cardiac myocytes respond to stress signals by hypertrophic growth and activation of a fetal gene program. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy, and mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals. To further define the roles of HDACs in cardiac hypertrophy, we analyzed the effects of HDAC inhibitors on the responsiveness of primary cardiomyocytes to hypertrophic agonists. Paradoxically, HDAC inhibitors imposed a dose-dependent blockade to hypertrophy and fetal gene activation. We conclude that distinct HDACs play positive or negative roles in the control of cardiomyocyte hypertrophy. HDAC inhibitors are currently being tested in clinical trials as anti-cancer agents. Our results suggest that these inhibitors may also hold promising clinical value as therapeutics for cardiac hypertrophy and heart failure.     

Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.     

Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.     

Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.     

Histone deacetylase inhibitors and malignant melanoma

The search for antimelanoma agents acting by terminal differentiation via the pigmentation pathway has so far been unsuccessful, in part because of tumor heterogeneity and loss of function of pigmentation genes. Some differentiation agents, however, have emerged as inhibitors of histone deacetylases (HDAC), with consequences for chromosome remodeling, cell cycle arrest and selective toxicity in cultured melanoma cells compared with normal melanocytes. Few effects have been found on pigmentation, except paradoxically the down-regulation of TRP-1. Of the many genes regulated by HDAC inhibitors, induction of p21(WAF1/Cip1) is the most consistent finding and is associated with G(1) or G(2) phase blocks. Some melanoma cell lines appear to lack an HDAC inhibitor-specific G(2) checkpoint and viability is thus compromised by dividing with inappropriately-modified chromatin. Most cultured melanoma cells undergo apoptosis following treatment with HDAC inhibitors, via a mitochondrial and caspase-dependent pathway. However, the molecular mechanism may vary with cell line and HDAC inhibitor class. Tumor selectivity cannot yet be attributed to specific types or levels of HDACs, nor has the possibility of acetylation of non-histone targets been excluded. Elucidation of these complexities may be rewarding, in terms of directing the multiple consequences of inhibiting histone deacetylation towards overcoming the therapeutic problems of melanoma heterogeneity and emergence of resistance. Success in the clinic may require combination with agents that synergize with the cell cycle blocking and pro-apoptotic action of HDAC inhibitors.