Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.     

Inhibition of histone deacetylase causes emphysema

In patients with chronic obstructive pulmonary disease (COPD), histone deacetylase (HDAC) expression and activity are reduced in the lung tissue. However, whether HDAC activity controls the maintenance of the lung alveolar septal structures has not been investigated. To explore the consequences of HDAC inhibition and address the question of whether HDAC inhibition causes lung cell apoptosis and emphysema, male Sprague-Dawley rats and human pulmonary microvascular endothelial cells (HPMVEC) were treated with trichostatin A (TSA), a specific inhibitor of HDACs. Chronic TSA treatment increased the alveolar air space area, mean linear intercept, and the number of caspase-3-positive cells in rat lungs. TSA suppressed hypoxia-inducible factor-1α (HIF-1α), VEGF, and lysyl oxidase (LOX) and increased microtubule-associated protein-1 light chain 3 (LC3), p53, and miR34a microRNA expression in both rat lungs and cultured HPMVEC. Gene silencing of HDAC2 using small interfering RNA (siRNA) in cultured HPMVEC resulted in the suppression of HIF-1α, VEGF, and LOX and an increase of p53 expression. These data indicate that HDAC inhibition causes emphysema and that HDAC-dependent mechanisms contribute to the maintenance of the adult lung structure. Our results also suggest that the increase in apoptosis, as a consequence of HDAC inhibition, is associated with decreased VEGF and HIF-1α expression.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.     

Epstein-Barr virus-infected Akata cells are sensitive to histone deacetylase inhibitor TSA-provoked apoptosis

Epstein-Barr virus (EBV) infects more than 90 % of the world's population and has a potential oncogenic nature. A histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), has shown potential ability in cancer chemoprevention and treatment, but its effect on EBV-infected Akata cells has not been examined. This study investigated the effect of TSA on the proliferation and apoptosis of the cells. TSA inhibited cell growth and induced cytotoxicity in the EBV-infected Akata cells. TSA treatment sensitively induced apoptosis in the cell, which was demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to the sub-G0/G1 phase in flow cytometric analysis, and the ladder formation of genomic DNA. Western blot analysis showed that caspase-dependent pathways are involved in the TSA-induced apoptosis of EBV-infected Akata cells. Overall, this study shows that EBV-infected B lymphomas are quite sensitive to TSA-provoked apoptosis.     

Dynamic Rearrangement of F-Actin Is Required to Maintain the Antitumor Effect of Trichostatin A

Actin plays a role in various processes in eukaryotic cells, including cell growth and death. We investigated whether the antitumor effect of trichostatin A (TSA) is associated with the dynamic rearrangement of F-actin. TSA is an antitumor drug that induces hyper-acetylation of histones by inhibiting histone deacetylase. HeLa human cervical cancer cells were used to measure the antitumor effect of TSA. The percent cell survival was determined by an MTT assay. Hypodiploid cell formation was assessed by flow cytometry. Collapse of the mitochondrial membrane potential (MMP) was identified by a decrease in the percentage of cells with red MitoProbe J-aggregate (JC-1) fluorescence. Cell survival was reduced by treatment with TSA, as judged by an MTT assay and staining with propidium iodide, FITC-labeled annexin V, or 4′,6-diamidino-2-phenylindole (DAPI). TSA also induced an MMP collapse, as judged by the measurement of intracellular red JC-1 fluorescence. In addition, the F-actin depolymerizers cytochalasin D (CytoD) and latrunculin B (LatB) induced an MMP collapse and increased apoptotic cell death in HeLa cells. However, our data show that apoptotic cell death and the MMP collapse induced by TSA were decreased by the co-treatment of cells with CytoD and LatB. These findings demonstrate that the dynamic rearrangement of F-actin might be necessary for TSA-induced HeLa cell apoptosis involving a TSA-induced MMP collapse. They also suggest that actin cytoskeleton dynamics play an important role in maintaining the therapeutic effects of antitumor agents in tumor cells. They further suggest that maintaining the MMP could be a novel strategy for increasing drug sensitivity in TSA-treated tumors.     

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 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.     

Caspase-independent death of human osteosarcoma cells by flavonoids is driven by p53-mediated mitochondrial stress and nuclear translocation of AIF and endonuclease G

Flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine that has long been used in Korea as both a food additive and antitumor agent. It was previous reported that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), inhibited the proliferation and induced apoptosis in human osteosarcoma (HOS) cells. This study examined the mechanisms involved in the RCMF-mediated apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis in HOS cells by inducing p53 in the cells resulting in the decrease in Bcl-2 level, activation of Bax, and cytoplasmic release of cytochrome c, which led to the translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus. However, the RCMF-induced apoptosis was suppressed by transfecting the cells with antisense p53 oligonucleotides but not by treating them with a MAPK or caspase inhibitor. This suppression occurred through the regulation of Bcl-2 members as well as by preventing the nuclear translocation of the mitochondrial apoptogenic factors. Overall, it appears that p53-mediated mitochondrial stress and the nuclear translocation of AIF and EndoG are mainly required for the apoptosis induced by RCMF.     

Sensitization of osteosarcoma cells to death receptor-mediated apoptosis by HDAC inhibitors through downregulation of cellular FLIP

Fas-mediated apoptosis plays an important role in elimination of tumor cells in vivo, but some tumor-derived cells are resistant to this mechanism. Here, we show that treatment with the histone deacetylase (HDAC) inhibitor FR901228 renders Fas-resistant osteosarcoma cell lines sensitive to Fas-mediated apoptosis by downregulating expression of cellular FLIP (cellular FLICE-inhibitory protein), an inhibitor of Fas-mediated activation of caspase-8. Moreover, sensitization to Fas-mediated apoptosis was also induced in Fas-resistant osteosarcoma cells by suppressing FLIP expression using FLIP-specific RNA interference. HDAC inhibitors including FR901228 were shown to induce downregulation of cellular FLIP through inhibiting generation of FLIP mRNA, rather than stimulating degradation at either protein or mRNA level, and the inhibition was independent of de novo protein synthesis. These results clearly indicate that some tumor cells exhibit a phenotype resistant to death receptor-mediated apoptosis by expressing cellular FLIP, and that HDAC inhibitors sensitize such resistant tumor cells by directly downregulating cellular FLIP mRNA.     

Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.