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.     

Ellagic acid inhibits adipocyte differentiation through coactivator-associated arginine methyltransferase 1-mediated chromatin modification

Chromatin remodeling is a key mechanism in adipocyte differentiation. However, it is unknown whether dietary polyphenols are epigenetic effectors for adiposity control. Ellagic acid (EA) is a naturally occurring polyphenol in numerous fruits and vegetables. Recently, EA-containing foods have been reported to reduce adiposity. In the present study, we sought to determine whether EA inhibits adipogenesis by modifying chromatin remodeling in human adipogenic stem cells (hASCs). qPCR microarray of chromatin modification enzymes revealed that 10 μmol/L of EA significantly inhibits histone deacetylase (HDAC)9 down-regulation. In addition, EA was associated with up-regulation of HDAC activity and a marked reduction of histone acetylation levels. However, chemical inhibition of HDAC activity or depletion of HDAC9 by siRNA were not sufficient to reverse the antiadipogenic effects of EA. Intriguingly, EA treatment was also associated with reduced histone 3 arginine 17 methylation levels (H3R17me2), implying the inhibitory role of EA in coactivator-associated arginine methyltransferase 1 (CARM)1 activity during adipogenesis. Boosting CARM1 activity by delivering cell-penetrating peptides of CARM1 not only recovered H3R17me2 but also restored adipogenesis evidenced by H3 acetylation at lysine 9, HDAC9 down-regulation, PPARγ expression and triglyceride accumulation. Taken together, our data suggest that reduced CARM1 activity by EA results in a decrease of H3R17me2 levels, which may interrupt consecutive histone remodeling steps for adipocyte differentiation including histone acetylation and HDAC9 dissociation from chromatin. Our work provides the mechanistic insights into how EA, a polyphenol ubiquitously found in fruits and vegetables, attenuates human adipocyte differentiation by altering chromatin remodeling.     

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.     

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.     

Activation of Mitochondrial Pathway is Crucial for Tumor Selective Induction of Apoptosis by LAQ824

HDAC inhibitors are promising antitumor drugs with several HDAC inhibitors already in clinical trials. LAQ824, a potent pan-HDAC inhibitor, has been shown to induce cell cycle arrest and cell death. However, the mechanism of its antitumor effects and specially its tumor selectivity are still poorly understood. The focus of this study is to elucidate LAQ824 mediated anti-proliferative effects in lung carcinoma cells and the mechanism underlying the different sensitivity of LAQ824 to cancer and normal cells. In this study, LAQ824 mediated apoptosis was found to occur mainly via activation of the mitochondrial death pathway by inducing Apaf1 and caspase 9 and promoting mitochondrial release of key proapoptotic factors in lung cancer cells, but not in normal fibroblast cells. Using chromatin immunoprecipitation assay, we found that RNA PolII binding and histone H3 acetylation levels at Apaf1 promoter were increased following LAQ824 treatment, explaining LAQ824 induced expression of Apaf1 in lung cancer cells. Furthermore, we showed that LAQ824 only triggered the release of mitochondrial proapoptotic factors such as cytochrome C (Cyto C) and apoptosis inducing factor (AIF) in lung cancer cells but not in normal blast cells. In addition, LAQ824 was found to induce Bax translocation in lung cancer cell, which may play important role in the induction of the release of mitochondrial proapoptotic factors. These data provide insight into the mechanism underlying the selective induction of apoptosis by LAQ824 in cancer cells.     

Histone acetyltransferase p300 regulates the expression of human pituitary tumor transforming gene (hPTTG)

The human pituitary tumor transforming gene (hPTTG) serves as a marker for malignancy grading in several cancers, hPTTG is in volved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase (HAT) p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation (ChIP)analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found thattreatment of 293T cells with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylation modification in the regulation of hPTTG.     

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.     

Aza-PBHA,a potent histone deacetylase inhibitor,inhibits human gastric-cancer cell migration via PKCα-mediated AHR-HDAC interactions

Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.     

Cu2+ is required for pyrrolidine dithiocarbamate to inhibit histone acetylation and induce human leukemia cell apoptosis

Pyrrolidine dithiocarbamate (PDTC) has been considered as a potential anticancer drug due to its powerful apoptogenic effect towards cancer cells, where Cu(2+) plays a distinct yet undefined role. Here we report that Cu(2+) is critically needed for PDTC to inhibit histone acetylation in both human leukemia HL-60 cells and human hepatoma Hep3B cells. The inhibition of histone acetylation mainly resulted from the increase of intracellular Cu(2+), but was not due to the inhibition of NF-kappaB activity by PDTC-Cu(2+) since the combinations of Cu(2+) with SN50, MG132 (two known NF-kappaB inhibitors), or bathocuproine disulfonate (BCS, a specific Cu(2+) chelator that does not cross the plasma membrane), did not lead to obvious inhibition of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cells exposed to PDTC-Cu(2+) showed a comparable decrease in histone acetylation levels in HL-60 cells in the absence or presence of the HDAC inhibitors, trichostatin A (TSA) or sodium butyrate (NaBu); the inhibition rates were about 45, 44 and 43%, respectively. PDTC-Cu(2+) had no effect on the activity of HDAC in vitro, but significantly inhibited the HAT activity both in HL-60 cells and in a cell-free in vitro system. PDTC-Cu(2+) also induced HL-60 cell apoptosis, and treating cells with TSA, NaBu or BCS significantly attenuated the apoptosis induced by PDTC-Cu(2+). Collectively, these results showed that inhibition of histone acetylation represents a distinct mechanism for the cytotoxicity of PDTC in the presence of Cu(2+), where HAT is its possible molecular target.     

Histone hyperacetylation is associated with amelioration of experimental colitis in mice

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.