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.     

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc.Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range.In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC’s) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX).Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.     

Identification of KD5170: a novel mercaptoketone-based histone deacetylase inhibitor

We report the identification of KD5170, a potent mercaptoketone-based Class I and II-histone deacetylase inhibitor that demonstrates broad spectrum cytotoxic activity against a range of human tumor-derived cell lines. KD5170 exhibits robust and sustained histone H3 hyperacetylation in HCT-116 xenograft tumors following single oral or i.v. dose and inhibition of tumor growth following chronic dosing.     

Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography-electrospray mass spectrometry

A rapid method was developed for the quantitative determination of the novel histone deacetylase inhibitor, MS-275, in human plasma. Calibration curves were constructed in the range of 1-100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step protein precipitation with acetonitrile of 0.1 ml samples. The analysis was performed on a column (75 mm x 4.6 mm i.d.) packed with 3.5 microm Phenyl-SB material, using methanol-10mM ammonium formate (55:45 (v/v)) as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always < or =5.58 and <11.4% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of MS-275 in a cancer patient.     

Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)

UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.     

Alleviation of osteoarthritis by Trichostatin A, a histone deacetylase inhibitor, in experimental osteoarthritis

This study investigated the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on cartilage degradation in an experimental model of osteoarthritis (OA). Thirty-two male New Zealand rabbits underwent unilateral anterior cruciate ligament transection (ACLT) on left knee joints to induce OA and were randomly divided into two groups (n = 16), the TSA group was injected intra-articularly with 0.3 ml TSA [250 ng/ml in the dimethylsulphoxide (DMSO)], the OA group received DSMO since 4 weeks after operation once a week for 5 weeks. Rabbits were killed seven days after the last injection. Left knee cartilage was harvested for morphological, histological and genetic analysis. Another ten rabbits were used for normal control and received no injection. The TSA group showed less cartilage degradation as compared to the OA group assessed by morphological and histological evaluation. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and interleukin-1 (IL-1) was increased significantly in the OA group compared to the normal group. The elevated expression was reduced by TSA. Our results suggest that TSA could be considered as a potential agent for treatment for OA.     

Mechanism of histone deacetylase inhibitor Trichostatin A induced apoptosis in human osteosarcoma cells

Although histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in malignancy, how they exert their effect on osteosarcoama cells is as yet unclear. This study was undertaken to investigate the underlying mechanism of a HDAC inhibitor Trichostatin A (TSA)-induced apoptosis in a osteosarcoma cell line HOS. We observed that TSA treatment decreased the viability of the cells and prominently increased acetylation of histone H3. Evidence was obtained indicating that TSA induced apoptosis of HOS cells as follows: (1) Generation of DNA fragmentation; (2) activation of procaspase-3; (3) cleavage of PARP; and (4) increase of DNA hypoploidy. The reduction of MMP and the release of cytochrome c to cytosol were also shown, indicating that TSA induces apoptosis in HOS cells in a histone acetylation- and mitochondria-dependent fashions. We also examined whether TSA can sensitize HOS cells to the action of an antitumor agent genistein. The combination therapy of TSA and genistein showed synergistic anticancer effect indicating that TSA can be considered as a novel therapeutic strategy for osteosarcoma not only from its direct apoptosis-inducing activity but also from the possibility of sensitization to other antitumor agents.     

Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.     

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury

Cardiac injury is a major complication for oxidative-stress-generating anticancer agents exemplified by Adriamycin (ADR). Recently, several histone deacetylase inhibitors (HDACIs) including phenylbutyrate (PBA) have shown promise in the treatment of cancer with little known toxicity to normal tissues. PBA has been shown to protect against oxidative stress in normal tissues. Here, we examined whether PBA might protect heart against ADR toxicity in a mouse model. The mice were i.p. injected with ADR (20 mg/kg). PBA (400 mg/kg/day) was i.p. injected 1 day before and daily after the ADR injection for 2 days. We found that PBA significantly decreased the ADR-associated elevation of serum lactate dehydrogenase and creatine kinase activities and diminished ADR-induced ultrastructual damages of cardiac tissue by more than 70%. Importantly, PBA completely rescued ADR-caused reduction of cardiac functions exemplified by ejection fraction and fraction shortening, and increased cardiac manganese superoxide dismutase (MnSOD) protein and activity. Our results reveal a previously unrecognized role of HDACIs in protecting against ADR-induced cardiac injury and suggest that PBA may exert its cardioprotective effect, in part, by the increase of MnSOD. Thus, combining HDACIs with ADR could add a new mechanism to fight cancer while simultaneously decrease ADR-induced cardiotoxicity.     

Histone deacetylase inhibitor,AR-42, exerts antitumor effects by inducing apoptosis and cell cycle arrest in Y79 cells

Retinoblastoma (RB) is the most common type of intraocular malignant tumor that occurs in childhood. AR-42, a member of a newly discovered class of phenylbutyrate-derived histone deacetylase inhibitors, exerts antitumor effects on many cancers. In the present study, we initially evaluated the effect of AR-42 towards RB cells and explored the underlying mechanism in this disease. Our results found that AR-42 showed powerful antitumor effects at low micromolar concentrations by inhibiting cell viability, blocking cell cycle, stimulating apoptosis in vitro, and suppressing RB growth in a mouse subcutaneous tumor xenograft model. Furthermore, the AKT/nuclear factor-kappa B signaling pathway was disrupted in Y79 cells treated with AR-42. In conclusion, we propose that AR-42 might be a promising drug treatment for RB.     

CS055 (Chidamide/HBI-8000), a novel histone deacetylase inhibitor, induces G1 arrest, ROS-dependent apoptosis and differentiation in human leukaemia cells

CS055 (Chidamide/HBI-8000) is a novel benzamide-type HDACi (histone deacetylase inhibitor), which has entered Phase I clinical trials in the U.S. and Phase II/III in China. In the present study, we investigated the effects of CS055 on proliferation, differentiation and apoptosis in human leukaemia cell lines and primary myeloid leukaemia cells. The results showed that at low concentrations (<1?μM), CS055 induced G1 arrest. At moderate concentrations (0.5?μM-2?μM), CS055 induced differentiation, as determined by the increased expression of the myeloid differentiation marker CD11b. At relatively high concentrations (2?μM-4?μM), CS055 potently induced caspase-dependent apoptosis. Co-treatment with the ROS (reactive oxygen species) scavengers N-acetyl-L-cysteine or Tiron blocked CS055-induced cell differentiation and apoptosis, suggesting an essential role for ROS in these effects. Cytochrome c release and ROS-mediated mitochondrial dysfunction are involved in CS055-induced apoptosis of leukaemia. In addition to cell lines, CS055 also exhibits therapeutic effects in human primary leukaemia cells. Moreover, daily oral CS055 treatment of nude mice bearing HL60 cell xenografts suppressed tumour growth, induced tumour cell apoptosis and prolonged the survival of tumour-bearing mice. In conclusion, our findings demonstrate that CS055 is a novel HDACi with potential chemotherapeutic value in several haematological malignancies, especially leukaemia.     

Bim plays a crucial role in synergistic induction of apoptosis by the histone deacetylase inhibitor SBHA and TRAIL in melanoma cells

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor – induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X_L, Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim_EL. Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma. This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council, Australia. X.D. Zhang is a Cancer Institute NSW Fellow.     

Identification of a potent non-hydroxamate histone deacetylase inhibitor by mechanism-based drug design

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, we synthesized several suberoylanilide hydroxamic acid (SAHA)-based compounds designed on the basis of the catalytic mechanism of HDACs. Among these compounds, 5b was found to be as potent as SAHA. Kinetic enzyme assays and molecular modeling suggested that the mercaptoacetamide moiety of 5b interacts with the zinc in the active site of HDACs and removes a water molecule from the reactive site of the deacetylation.     

Design,synthesis and evaluation of novel indirubin-based N-hydroxybenzamides,N-hydroxypropenamides and N-hydroxyheptanamides as histone deacetylase inhibitors and antitumor agents

Several novel indirubin-based N-hydroxybenzamides, N-hydropropenamides and N-hydroxyheptanamides (4a-h, 7a-h, 10a-h) were designed using a fragment-based approach with structural features extracted from several previously reported HDAC inhibitors, such as SAHA (vorinostat), MGCD0103 (mocetinostat), nexturastat A and PXD-101 (belinostat). The biological results reveal that our compounds showed excellent cytotoxicity toward three common human cancer cell lines (SW620, PC-3 and NCI-H23) with IC₅₀ values ranging from 0.09 to 0.007 µM. The cytotoxicity of the compounds was equipotent or even up to 10-times more potent than adriamycin and up to 205-times more potent than SAHA. Among the series of N-hydroxypropenamides, compounds 10a-d were the most potent HDAC inhibitors as well as cytotoxicity toward the cell lines tested. In addition, the strong inhibitory activites toward HDAC of our compounds were observed with IC₅₀ values of below-micromolar range. Especially, compound 4a inhibited HDAC6 with an IC₅₀ value of 29-fold lower than that against HDAC2 isoform. Representative compounds 4a and 7a were found to significantly arrest SW620 cells at G0/G1 phase. Compounds 7a and 10a were found to strongly induce apoptosis in SW620 cells. Docking studies revealed some important features affecting the selectivity against HDAC6 isoform. The results clearly demonstrate the potential of the indirubin-hydroxamic acid hybrids and these compounds should be very promising for further development.     

Apicidin, a histone deacetylase inhibitor, induces apoptosis and Fas/Fas ligand expression in human acute promyelocytic leukemia cells.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21(WAF1/Cip1). In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.