Search

Background Interleukin-8 (IL-8) and heat shock protein 60 (Hsp60) play crucial roles in cell survival and maintenance of cellular homoeostasis. However, cross talks between these two proteins are not defined. Methods IL-8 expression in tumour tissue sections was analysed by immunohistochemistry. IL-8 expression and release in cancer cells was quantified using enzyme-linked immunosorbent assay (ELISA). Apoptosis was quantified using caspase activity and Annexin-V/PI staining. Results We observed IL-8 release from cancer cells in response to histone deacetylase inhibitor, apicidin (Api), and non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, thapsigargin (TG). IL-8 release was increased upon TG-treatment. TG-induced IL-8 expression was reduced in the presence of Api in Bax-dependent manner. Increased apoptosis was associated with decreased IL-8 expression in response to combined treatment of TG and Api. TG and Api combination induced caspase-8 and caspase-9 dependent apoptosis. Hsp60 knockdown abrogated IL-8 expression induced by Api, TG, and their combination. The level of TGF-β, an upstream regulator of IL-8, was decreased upon Hsp60-silencing. Knocking down Hsp60 decreased IL-8 expression and its release in prostate cancer cell xenograft tumours in SCID mice. Conclusion This study describes the underlying mechanism associated with apoptosis resistance mediated via Hsp60-IL-8 axis in cancer.

MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.

Background Nearly half of the world’s population (3.2 billion people) were at risk of malaria in 2015, and resistance to current therapies is a major concern. While the standard of care includes drug combinations, there is a pressing need to identify new combinations that can bypass current resistance mechanisms. In the work presented here, a combined transcriptional drug repositioning/discovery and machine learning approach is proposed. Methods The integrated approach utilizes gene expression data from patient-derived samples, in combination with large-scale anti-malarial combination screening data, to predict synergistic compound combinations for three Plasmodium falciparum strains (3D7, DD2 and HB3). Both single compounds and combinations predicted to be active were prospectively tested in experiment. Results One of the predicted single agents, apicidin, was active with the AC50 values of 74.9, 84.1 and 74.9 nM in 3D7, DD2 and HB3 P. falciparum strains while its maximal safe plasma concentration in human is 547.6 ± 136.6 nM. Apicidin at the safe dose of 500 nM kills on average 97% of the parasite. The synergy prediction algorithm exhibited overall precision and recall of 83.5 and 65.1% for mild-to-strong, 48.8 and 75.5% for moderate-to-strong and 12.0 and 62.7% for strong synergies. Some of the prospectively predicted combinations, such as tacrolimus-hydroxyzine and raloxifene-thioridazine, exhibited significant synergy across the three P. falciparum strains included in the study. Conclusions Systematic approaches can play an important role in accelerating discovering novel combinational therapies for malaria as it enables selecting novel synergistic compound pairs in a more informed and cost-effective manner. Electronic supplementary material The online version of this article (10.1186/s12936-018-2294-5) contains supplementary material, which is available to authorized users.

Despite rapid advances in the diagnostic and surgical procedures, hepatocellular carcinoma (HCC) remains one of the most difficult human malignancies to treat. This may be due to the chemoresistant behaviors of HCC. It is believed that acquired resistance could be overcome and improve the overall survival of HCC patients by understanding the mechanisms of chemoresistance in HCC. A stable HA22T cancer line, which is chronically resistant to a histone deacetylase inhibitor, was established. After comparing the molecular mechanism of apicidin-R HA22T cells to parental ones by Western blotting, cell cycle-regulated proteins did not change in apicidin-R cells, but apicidin-R cells were more proliferative and had higher tumor growth (wound-healing assay and nude mice xenograft model). Moreover, apicidin-R cells displayed increased levels of p-IGF-IR, p-PI3K, p-Akt, Bcl-xL, and Bcl-2 but also significantly inhibited the tumor suppressor PTEN protein and apoptotic pathways when compared to the parental strain. Therefore, the highly proliferative effect of apicidin-R HA22T cells was blocked by Akt knockdown. For all these findings, we believe that novel strategies to attenuate IGF-IR/PI3K/Akt signaling could overcome chemoresistance toward the improvement of overall survival of HCC patients.

Apicidin, a fungal metabolite that functions as a histone deacetylase inhibitor, induces apoptosis in cancer cells. We investigated the molecular mechanisms of the anti-cancer effects of apicidin in mouse Neuro-2a neuroblastoma cells. Apicidin induced apoptotic cell death and activation of caspase-12, -9, and -3. Apicidin induced expression of endoplasmic reticulum (ER) stress-associated proteins, including CCAAT/enhancer binding protein homologous protein (CHOP), cleavage of activating transcription factor 6α, and phosphorylation of eukaryotic initiation factor 2α. Inhibition of ER stress by CHOP knockdown or using the ER stress inhibitors, salubrinal and 4-phenylbutyric acid, reduced apicidin-induced cell death. Apicidin induced reactive oxygen species accumulation and mitochondrial membrane potential loss. An antioxidant, N-acetyl cysteine, reduced apicidin-induced cell death, CHOP expression, and mitochondrial dysfunction. In addition, apicidin increased cytosolic Ca(2+), which was blocked by 2-aminoethoxydiphenyl borate, an antagonist of inositol 1,4,5-trisphosphate receptor, and BAPTA-AM, an intracellular Ca(2+) chelator. 2-Aminoethoxydiphenyl borate and BAPTA-AM inhibited apicidin-induced cell death and ER stress. Interestingly, apicidin induced phosphorylation of phospholipase Cγ1 (PLCγ1) and epidermal growth factor receptor (EGFR), and inhibition of PLCγ1 and EGFR reduced cell death and ER stress. Finally, apicidin-induced histone H3 hyperacetylation and reduction of histone deacetylase 2 mRNA expression were not affected by either a PLCγ1 inhibitor, U73122, or the antioxidant, N-acetyl cysteine. Taken together, the results suggest that apicidin induces apoptosis by ER stress and mitochondrial dysfunction via PLCγ1 activation, Ca(2+) release, and reactive oxygen species accumulation in Neuro-2a neuroblastoma cells.

Background: Over the last decade, several drugs that inhibit class I and/or class II histone deacetylases (HDACs) have been identified, including trichostatin A, the cyclic depsipeptide FR901228 and the antibiotic apicidin. These compounds have had immediate application in cancer research because of their ability to reactivate aberrantly silenced tumour suppressor genes and/or block tumour cell growth. Although a number of HDAC inhibitors are being evaluated in preclinical cancer models and in clinical trials, little is known about the differences in their specific mechanism of action and about the unique determinants of cancer cell sensitivity to each of these inhibitors. Methods: Using a combination of cell viability assays, HDAC enzyme activity measurements, western blots for histone modifications, microarray gene expression analysis and qRT–PCR, we have characterised differences in trichostatin A vs depsipeptide-induced phenotypes in lung cancer, breast cancer and skin cancer cells and in normal cells and have then expanded these studies to other HDAC inhibitors. Results: Cell viability profiles across panels of lung cancer, breast cancer and melanoma cell lines showed distinct sensitivities to the pan-inhibitor TSA compared with the class 1 selective inhibitor depsipeptide. In several instances, the cell lines most sensitive to one inhibitor were most resistant to the other inhibitor, demonstrating these drugs act on at least some non-overlapping cellular targets. These differences were not explained by the HDAC selectivity of these inhibitors alone since apicidin, which is a class 1 selective compound similar to depsipeptide, also showed a unique drug sensitivity profile of its own. TSA had greater specificity for cancer vs normal cells compared with other HDAC inhibitors. In addition, at concentrations that blocked cancer cell viability, TSA effectively inhibited purified recombinant HDACs 1, 2 and 5 and moderately inhibited HDAC8, while depsipeptide did not inhibit the activity of purified HDACs in vitro but did in cellular extracts, suggesting a potentially indirect action of this drug. Although both depsipeptide and TSA increased levels of histone acetylation in cancer cells, only depsipeptide decreased global levels of transcriptionally repressive histone methylation marks. Analysis of gene expression profiles of an isogenic cell line pair that showed discrepant sensitivity to depsipeptide, suggested that resistance to this inhibitor may be mediated by increased expression of multidrug resistance genes triggered by exposure to chemotherapy as was confirmed by verapamil studies. Conclusion: Although generally thought to have similar activities, the HDAC modulators trichostatin A and depsipeptide demonstrated distinct phenotypes in the inhibition of cancer cell viability and of HDAC activity, in their selectivity for cancer vs normal cells, and in their effects on histone modifications. These differences in mode of action may bear on the future therapeutic and research application of these inhibitors.

The objectives of this study were to develop physiologically based models for the pharmacokinetics (PK) and organ distribution of apicidin in rats and mice and to predict human PK in blood and organs. The PK of apicidin was characterized in rats and mice after i.v. bolus injection, and distribution to various tissues was determined in rats following i.v. infusions at steady state. The developed models were prospectively validated within rat and within mouse and by scaling from rat to mouse using data after multiple i.v. injections. Human PK was predicted by the physiologically based modeling using intrinsic clearance data for humans from in vitro experiments. The Cls predicted for human (9.8 ml/min/kg) was lower than those found in mice (116.9 ml/min/kg) and rats (61.6 ml/min/kg), and the Vss predicted for human (1.9 l/kg) was less than in mice (2.0 l/kg) and rats (2.5 l/kg). Consequently, the predicted t 1/2 was longer in human (2.3 h) than in mice and rats (0.4 and 0.9 h, respectively). The highest concentrations of apicidin were predicted in liver followed by adipose tissue, kidney, lung, spleen, heart, arterial blood, venous blood, small intestine, stomach, muscle, testis, and brain. The developed models adequately described the PK of apicidin in rats and mice and were applied to predict human PK. These models may be useful in predicting human blood and tissue concentrations of apicidin under different exposure conditions.

Nutlin-3, a small-molecule MDM2 inhibitor, restores p53 function and is, thus, an appealing candidate for the treatment of cancers retaining wild-type p53. However, nutlin-3 applied as single agent may be insufficient for cancer therapy. Therefore, we explored whether the anticancer activity of nutlin-3 could be enhanced by combination with histone deacetylase inhibitors (HDACi), i.e. vorinostat, sodium butyrate, MS-275 and apicidin. We found that nutlin-3 and HDACi cooperated to induce cell death in the p53 wild-type cell lines A549 and A2780, but not in the p53 null cell line PC-3, as assessed by Alamar Blue assay and flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization. Combination index analysis showed that the effect was synergistic. For comparison, we tested nutlin-3 in combination with paclitaxel, revealing that nutlin-3 antagonized the cytotoxic activity of paclitaxel. To shed light on the underlying mechanism of the synergistic action of nutlin-3 and HDACi, we determined the acetylation status of p53 by immunoblotting and the mRNA levels of MDM2 and MDM4 by real-time RT-PCR. We observed vorinostat to induce p53 hyperacetylation, to reduce the constitutive gene expression of MDM2 and MDM4, and to counteract the nutlin-3-induced upregulation of MDM2 gene expression. In conclusion, our study shows that HDACi amplify the antitumor activity of nutlin-3-possibly by inducing p53 hyperacetylation and/or MDM2 and/or MDM4 downregulation-suggesting that treatment with a combination of nutlin-3 and HDACi may be an effective strategy for treating tumors with wild-type p53.

SD-2007 is a new derivative of apicidin, an anti-parasitic agent and a histone deacetylase (HDAC) inhibitor. A subacute toxicological evaluation of SD-2007 was investigated for 2 weeks in ICR mice. After oral administration of SD-2007 (0, 0.2, 1, 5 or 25 mg/mouse), the clinical signs, mortalities, body weight changes, blood biochemical parameters, absolute and relative organ weights were examined. One day after the administration of SD-2007, excretion of soft feces in 1 and 5 mg/head groups, and one male in 25 mg/mouse group developed diarrhea, but theses complications were disappeared two days after administration. No mortalities were observed in animals up to 25 mg/mouse (LD(50),25 mg/kg), but absolute and relative weights of testes were significantly lower at the highest dose group (25 mg/mouse) and serum LDH and glucose levels were elevated in male mice. In addition, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities were reduced in female mice at all dosages. These data suggest that SD- 2007 may be sex specific and be toxic to the male reproductive organ, and thus our findings require further investigation and in particular chronic toxicological investigations should be investigated.

In this study, we investigated the effects of various histone deacetylase (HDAC) inhibitors on adipocyte differentiation. Treatment of 3T3-L1 cells with HDAC inhibitors such as apicidin, trichostatin A, or suberoylanilide hydroxamic acid, under conditions that normally promote differentiation led to a dramatic attenuation of adipocyte differentiation. In contrast, sodium butyrate (NaB) treatment increased adipocyte differentiation. Accordingly, the expression of adipogenic marker genes such as FAS, aP2, PPARgamma, resistin, C/EBPalpha, ADD1/SREBP1c, and adiponectin were inhibited by apicidin treatment but not NaB, indicating that the adipocyte differentiation process could be differentially regulated depending on the type of HDAC inhibitor utilized. In addition, this differential effect seemed not to be due to disruption of the insulin- signaling pathway. Interestingly, our data showed that apicidin treatment could induce dedifferentiation of fully differentiated adipocytes, as evident by the fact that apicidin treatment led to a decrease of Oil Red O-stained adipocytes with a concomitant reduction in the expression levels of adipogenic marker genes. Collectively, our results suggest that adipocyte differentiation and dedifferentiation may be regulated by HDAC inhibitors.

Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.

We previously reported that the activation of p21(WAF1/Cip1) transcription by histone deacetylase inhibitor apicidin was mediated through Sp1 sites and pointed to the possible participation of protein kinase C (PKC). In this study, we investigated the role and identity of the specific isoforms of PKC involved and identified phosphatidylinositol 3-kinase (PI 3-kinase) as an upstream effector in HeLa cells. Using an isoform-specific pharmacological inhibitor of PKC, a PKC epsilon dominant-negative mutant, and antisense oligonucleotide to inhibit PKC epsilon specifically, we found that among PKC isoforms, PKC epsilon was required for the p21(WAF1/Cip1) expression by apicidin. In addition to PKC epsilon, PI 3-kinase appeared to participate in the activation of p21(WAF1/Cip1) promoter by apicidin, since inactivation of PI 3-kinase either by transient expression of dominant-negative mutant of PI 3-kinase or its specific inhibitors, LY294002 and wortmannin, attenuated the activation of p21(WAF1/Cip1) promoter and p21(WAF1/Cip1) protein expression by apicidin. Furthermore, membrane translocation of PKC epsilon in response to apicidin was blocked by the PI 3-kinase inhibitor, indicating the role of PI 3-kinase as an upstream molecule of PKC epsilon in the p21(WAF1/Cip1) promoter activation by apicidin. However, the p21(WAF1/Cip1) expression by apicidin appeared to be independent of the histone hyperacetylation, since apicidin-induced histone hyperacetylation of p21(WAF1/Cip1) promoter region was not affected by inhibition of PI 3-kinase and PKC, suggesting that the chromatin remodeling through the histone hyperacetylation alone might not be sufficient for the expression of p21(WAF1/Cip1) by apicidin. Taken together, these results suggest that the PI 3-kinase-PKC epsilon signaling pathway plays a pivotal role in the expression of the p21(WAF1/Cip1) by apicidin.

Background Granulocytes are important in the pathogenesis of several inflammatory diseases. Apoptosis is pivotal in the resolution of inflammation. Apoptosis in malignant cells is induced by histone deacetylase (HDAC) inhibitors, whereas HDAC inhibitors do not usually induce apoptosis in non-malignant cells. The aim of the present study was to explore the effects of HDAC inhibitors on apoptosis in human eosinophils and neutrophils. Methods Apoptosis was assessed by relative DNA fragmentation assay, annexin-V binding, and morphologic analysis. HDAC activity in nuclear extracts was measured with a nonisotopic assay. HDAC expression was measured by real-time PCR. Results A HDAC inhibitor Trichostatin A (TSA) induced apoptosis in the presence of survival-prolonging cytokines interleukin-5 and granulocyte-macrophage colony stimulating factor (GM-CSF) in eosinophils and neutrophils. TSA enhanced constitutive eosinophil and neutrophil apoptosis. Similar effects were seen with a structurally dissimilar HDAC inhibitor apicidin. TSA showed additive effect on the glucocorticoid-induced eosinophil apoptosis, but antagonized glucocorticoid-induced neutrophil survival. Eosinophils and neutrophils expressed all HDACs at the mRNA level except that HDAC5 and HDAC11 mRNA expression was very low in both cell types, HDAC8 mRNA was very low in neutrophils and HDAC9 mRNA low in eosinophils. TSA reduced eosinophil and neutrophil nuclear HDAC activities by ~50-60%, suggesting a non-histone target. However, TSA did not increase the acetylation of a non-histone target NF-κB p65. c-jun-N-terminal kinase and caspases 3 and 6 may be involved in the mechanism of TSA-induced apoptosis, whereas PI3-kinase and caspase 8 are not. Conclusions HDAC inhibitors enhance apoptosis in human eosinophils and neutrophils in the absence and presence of survival-prolonging cytokines and glucocorticoids.