Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.     

HDAC inhibitors potentiate the apoptotic effect of enzastaurin in lymphoma cells

Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphomas. We investigated the cytotoxicity and mechanisms of cell death of the combination of enzastaurin and low concentrations of histone deacetylase (HDAC) inhibitors in B-cell and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Combined enzastaurin/suberoylanilide hydroxamic acid treatment synergistically induced apoptosis in diffuse large B-cell lymphoma and T-cell lymphoma cell lines, and primary lymphoma/leukemia samples. Similarly, combined treatment of B-cell-like lymphoma cells with enzastaurin and two different HDAC inhibitors, valproic acid and (2E,4E)-6-(4-chlorophenylsulfanyl)-2,4-hexadienoic acid hydroxyamide synergistically induced apoptosis, suggesting the synergy is generalizable to other HDAC inhibitors. Our data indicate that enzastaurin/HDAC inhibitors therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancies and may be an effective therapeutic strategy. Potential mechanisms including enzastaurin mediated inhibition of HDAC inhibitor-induced compensatory survival pathways are discussed.     

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Inhibition of histone-deacetylase activity by short-chain fatty acids and some polyphenol metabolites formed in the colon

Colorectal cancer is the most abundant cause of cancer mortality in the Western world. Nutrition and the microbial flora are considered to have a marked influence on the risk of colorectal cancer, the formation of butyrate and other short-chain fatty acids (SCFAs) possibly playing a major role as chemopreventive products of microbial fermentation in the colon. In this study, we investigated the effects of butyrate, other SCFAs, and of a number of phenolic SCFA and trans-cinnamic acid derivatives formed during the intestinal degradation of polyphenolic constituents of fruits and vegetables on global histone deacetylase (HDAC) activity in nuclear extracts from colon carcinoma cell cultures using tert-butoxycarbonyl-lysine (acetylated)-4-amino-7-methylcoumarin (Boc-Lys(Ac)-AMC) as substrate. Inhibition of HDAC activity, e.g., by butyrate, is related to a suppression of malignant transformation and a stimulation of apoptosis of precancerous colonic cells. In nuclear extracts from HT-29 human colon carcinoma cells, butyrate was found to be the most potent HDAC inhibitor (IC(50)=0.09 mM), while other SCFAs such as propionate were less potent. In the same assay, p-coumaric acid (IC(50)=0.19 mM), 3-(4-OH-phenyl)-propionate (IC(50)=0.62 mM) and caffeic acid (IC(50)=0.85 mM) were the most potent HDAC inhibitors among the polyphenol metabolites tested. Interestingly, butyrate was also the most potent HDAC inhibitor in a whole-cell HeLa Mad 38-based reporter gene assay, while all polyphenol metabolites and all other SCFAs tested were much less potent; some were completely inactive. The findings suggest that butyrate plays an outstanding role as endogenous HDAC inhibitor in the colon, and that other SCFAs and HDAC-inhibitory polyphenol metabolites present in the colon seem to play a much smaller role, probably because of their limited levels, their marked cytotoxicity and/or their limited intracellular availability.     

HDAC inhibitors show differential epigenetic regulation and cell survival strategies on p53 mutant colon cancer cells

Besides inactivating tumour suppressor activity in cells, mutations in p53 confer significant oncogenic functions and promote metastasis and resistance to anticancer therapy. A variety of therapies involving genetic and epigenetic signalling events regulate tumorogenesis and progression in such cases. Pharmacological interventions with HDAC inhibitors have shown promise in therapy. This work explores the changes in efficacy of the four HDAC inhibitors SAHA, MS-275, valproic acid and sodium butyrate on a panel of colon cancer cell lines – HCT116 (p53 wt), HCT116 p53-/-, HT29 and SW480 (with mutations in p53). Clonogenic assays, gene profiling and epigenetic expression done on these cells point to p53 dependent differential activity of the 4 HDAC inhibitors which also elevate methylation levels in p53 mutant cell lines. In silico modelling establishes the alterations in interactions that lead to such differential activity of valproic acid, one of the inhibitors considered for the work. Molecular Dynamic simulations carried out on the valproic acid complex ensure stability of the complex. This work establishes a p53 dependent epigenetic signalling mechanism triggered by HDAC inhibition expanding the scope of HDAC inhibitors in adjuvant therapy for p53 mutant tumours.     

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non-toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an alpha-motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone-rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m-Carboxycinnamic acid bis-Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS-275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood-brain barrier, oral administration may allow deceleration of progressive alpha-motoneurone degeneration by epigenetic SMN2 gene activation.     

Histone deacetylase inhibitors for purging HIV-1 from the latent reservoir

A reservoir of latently infected memory CD4(+) T cells is believed to be the source of HIV-1 reemergence after discontinuation of antiretroviral therapy. HIV-1 eradication may depend on depletion of this reservoir. Integrated HIV-1 is inaccessible for expression, in part because of histone deacetylases (HDACs). One approach is to exploit the ability of HDAC inhibitors to induce HIV-1 expression from an integrated virus. With effective antiretroviral therapy, newly expressed HIV-1 is incapable of reinfecting naive cells. With HIV-1 expression, one assumes the infected cell dies and there is a progressive reduction in the size of the reservoir. The concept was tested using the HDAC inhibitor valproic acid. However, valproic acid is weak in inducing HIV-1 from latency in vitro. As such, clinical trials revealed a small or no effect on reducing the number of latently infected T cells in the peripheral blood. However, the new HDAC inhibitors vorinostat, belinostat and givinostat are more effective at targeting specific HDACs for HIV-1 expression than valproic acid. Here, we review studies on HDAC inhibitor-induced expression of latent HIV-1, with an emphasis on new and specific HDAC inhibitors. With increased potency for HIV-1 expression as well as safety and ease of oral administration, new HDAC inhibitors offer a unique opportunity to deplete the latent reservoir. An additional benefit is the antiinflammatory properties of HDAC inhibitors, including downregulation of HIV-1 coreceptor expression.     

Induction of GRP78 by valproic acid is dependent upon histone deacetylase inhibition

Valproic (2-propylpentanoic) acid is a commonly used drug in the treatment of bipolar disorder and epilepsy. The molecular mechanism that underlies its clinical efficacy remains controversial and is complicated by the broad range of intracellular effects of valproic acid, including its ability to inhibit histone deacetylase (HDAC) and induce protein chaperone expression. Here we show that an established HDAC inhibitor, trichostatin A, promotes ER chaperone expression in HEK293 cells. Furthermore, we use chemical derivatives of valproic acid to show that the ability to promote GRP78 levels directly correlates with the induction of histone H4 hyperacetylation. These results suggest that exposure to valproic acid enhances chaperone expression by a mechanism that involves histone hyperacetylation.     

Epigenetic histone modification by butyrate downregulates KIT and attenuates mast cell function

Short-chain fatty acid butyrate is produced from the bacterial fermentation of indigestible fiber in the intestinal lumen, and it has been shown to attenuate lung inflammation in murine asthma models. Mast cells (MCs) are initiators of inflammatory response to allergens, and they play an important role in asthma. MC survival and proliferation is regulated by its growth factor stem cell factor (SCF), which acts through the receptor, KIT. It has previously been shown that butyrate attenuates the activation of MCs by allergen stimulation. However, how butyrate mechanistically influences SCF signalling to impact MC function remains unknown. Here, we report that butyrate treatment triggered the modification of MC histones via butyrylation and acetylation, and inhibition of histone deacetylase (HDAC) activity. Further, butyrate treatment caused downregulation of SCF receptor KIT and associated phosphorylation, leading to significant attenuation of SCF-mediated MC proliferation, and pro-inflammatory cytokine secretion. Mechanistically, butyrate inhibited MC function by suppressing KIT and downstream p38 and Erk phosphorylation, and it mediated these effects via modification of histones, acting as an HDAC inhibitor and not via its traditional GPR41 (FFAR3) or GPR43 (FFAR2) butyrate receptors. In agreement, the pharmacological inhibition of Class I HDAC (HDAC1/3) mirrored butyrate's effects, suggesting that butyrate impacts MC function by HDAC1/3 inhibition. Taken together, butyrate epigenetically modifies histones and downregulates the SCF/KIT/p38/Erk signalling axis, leading to the attenuation of MC function, validating its ability to suppress MC-mediated inflammation. Therefore, butyrate supplementations could offer a potential treatment strategy for allergy and asthma via epigenetic alterations in MCs.     

Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat-shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up-regulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors – sodium butyrate, trichostatin A, and Class I HDAC-specific inhibitors MS-275 and apicidin, – all mimicked the ability of VPA to induce HSP70. Pre-treatment with phosphatidylinositol 3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the phosphatidylinositol 3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.