Suberoylanilide Hydroxamic Acid Treatment Research Articles

Histone Deacetylase Inhibitor SAHA Treatment Prevents the Development of Heart Failure after Myocardial Infarction via an Induction of Heat-Shock Proteins in Rats.

Protein quality control (PQC) in the heart plays an important role to maintain cellular protein homeostasis. Impairment of PQC may cause the development of heart failure. It is well known that histone deacetylase 6 (HDAC6) is an essential enzyme for regulating the cellular PQC response. In this study, we aimed at examining the association between HDAC6 and the chaperone system and the effects of HDAC6 inhibition in the development of heart failure following myocardial infarction (MI). MI was induced by coronary artery ligation. Coronary artery-ligated and sham-operated rats were divided into groups that were orally administered suberoylanilide hydroxamic acid (SAHA) or vehicle from the 2nd to 8th week after the operation. The cardiac function and protein expression levels in the viable left ventricle were analyzed by echocardiography, Western blotting, and immunohistochemistry at the 2nd and 8th weeks after the operation. The deacetylase activity of HDAC6 was markedly elevated during the development of heart failure after MI. In the failing heart, a decrease in heat-shock protein (HSP) contents and an accumulation of ubiquitinated proteins were observed, indicating PQC dysfunction. Inhibition of HDAC6 activity by SAHA treatment enhanced the translocation of heat-shock transcription factor 1 to the nucleus and induced the expression of HSP, resulting in maintenance of cellular protein homeostasis. The cardiac pump function after MI was also improved by SAHA administration. Our findings suggest that inhibition of HDAC6 activity is a novel approach for the treatment of heart failure following MI.

Overexpression of miR-4433 by suberoylanilide hydroxamic acid suppresses growth of CML cells and induces apoptosis through targeting Bcr-Abl.

Background: Targeting Bcr-Abl is the key for the treatment of CML. Although great progress has been achieved for the treatment of CML patients in chronic stage, effective drugs with good safety are not available for those in advanced stages of CML patients. In present study, a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was used to screen for microRNA that can target Bcr-Abl.Methods: RT-qPCR was used to determine Bcr-Abl and miR-4433 transcription level in CML cells. In CML cells, Proteins including PARP, caspase-3, acetyl-histone 3, histone 3 and Bcr-Abl, as well as Bcr-Abl downstream proteins were detected using western blot. Cell viability and apoptosis were monitored respectively by MTS assay and flow cytometry. The correlation between miR-4433 and Bcr-Abl was determined by luciferase reporter assay. The anti-tumor effect of miR-4433 to K562 cells was evaluated by nude mouse xenograft model in vivo.Results: SAHA up-regulated the acetylation level of histone 3, and effectively inhibited Bcr-Abl mRNA level and its downstream signal transduction pathway, while inhibiting the growth of CML cells and inducing apoptosis. Furthermore, bioinformatics tools predicted that miR-4433 is a putative microRNA targeting Bcr-Abl and that the expression level of miR-4433 was significantly increased after SAHA treatment in K562 cells. Luciferase activity analysis revealed that miR-4433 directly targets Bcr-Abl. Additionally, transient expression of miR-4433 abrogated Bcr-Abl activity and its downstream signaling pathways while inducing apoptosis in K562 cells. Moreover, stable expression of miR-4433 suppressed Bcr-Abl and its downstream signaling pathway, and inhibited the growth of K562 cells in vitro and the growth of K562-xenografts in nude mice.Conclusion: miR-4433 was identified as a microRNA targeting Bcr-Abl, which may be subject to epigenetic regulation of SAHA, a histone deacetylase inhibitor that has been approved by the US FDA for the treatment of cutaneous T-cell lymphoma. The findings of this study provide a molecular basis from another angle for the use of SAHA in the treatment of CML.

Suberoylanilide Hydroxamic Acid Triggers Autophagy by Influencing the mTOR Pathway in the Spinal Dorsal Horn in a Rat Neuropathic Pain Model.

Histone acetylation levels can be upregulated by treating cells with histone deacetylase inhibitors (HDACIs), which can induce autophagy. Autophagy flux in the spinal cord of rats following the left fifth lumber spinal nerve ligation (SNL) is involved in the progression of neuropathic pain. Suberoylanilide hydroxamic acid (SAHA), one of the HDACIs can interfere with the epigenetic process of histone acetylation, which has been shown to ease neuropathic pain. Recent research suggest that SAHA can stimulate autophagy via the mammalian target of rapamycin (mTOR) pathway in some types of cancer cells. However, little is known about the role of SAHA and autophagy in neuropathic pain after nerve injury. In the present study, we aim to investigate autophagy flux and the role of the mTOR pathway on spinal cells autophagy activation in neuropathic pain induced by SNL in rats that received SAHA treatment. Autophagy-related proteins and mTOR or its active form were assessed by using western blot, immunohistochemistry, double immunofluorescence staining and transmission electron microscopy (TEM). We found that SAHA decreased the paw mechanical withdrawal threshold (PMWT) of the lower compared with SNL. Autophagy flux was mainly disrupted in the astrocytes and neuronal cells of the spinal cord dorsal horn on postsurgical day 28 and was reversed by daily intrathecal injection of SAHA (n = 100nmol/day or n = 200nmol/day). SAHA also decreased mTOR and phosphorylated mTOR (p-mTOR) expression, especially p-mTOR expression in astrocytes and neuronal cells of the spinal dorsal horn. These results suggest that SAHA attenuates neuropathic pain and contributes to autophagy flux in astrocytes and neuronal cells of the spinal dorsal horn via the mTOR signaling pathway.

Disulfiram May Contribute to Apoptosis of Myeloma Cells By Reactivating of Cell Cycle Regulating Gene CDKN2A

Objective: Multiple myeloma (MM) is an incurable plasma-cell dyscrasia characterized by uncontrolled growth of malignant plasma cells in the bone marrow. Proteasome inhibitor bortezomib is a frontline MM drug, however acquired resistance to bortezomib remains a clinical hurdle. Disulfiram (tetraethylthiuram disulfide, DSF) belongs to promising cancer-killing drugs, and although the mechanism of its anticancer activity remains unclear, it has been suggested that this drug inhibits proteasome activity. Furthermore, disulfiram contains strong thio-reactive functional groups and since the catalytic mechanism of DNA methyltransferases (DNMTs) involves the covalent attack at the C6 position of cytosine by the thiol group of the catalytic cysteine on the DNMT enzyme, the DSF can function as DNMT non-nucleoside inhibitor with possible gene demethylation´s effect. On the other hand, DNMT inhibitors, 5-azacytidine (AZA) and 5-aza-2´-deoxycytidine (DAC) are nucleoside analogs, whose mechanism of action involves incorporation of the aza-modified base into DNA during DNA synthesis with subsequent covalent trapping of the DNMT enzyme. Therefore, these nucleoside analogs (AZA and DAC) can have significant cytotoxicity and can lead to major adverse effects, including myelosuppression, when administered to patients.Methods: RPMI8226 and U266 myeloma cells were exposed to DSF (0, 1 µM and 1 µM), DAC (0, 5 µM, 3, 75 µM and 5 µM), JNJ-7706621 (0, 5 µM and 5 µM) and 5 µM SAHA (suberoylanilide hydroxamic acid). Subsequently, we assessed for: i/ cell viability assay using 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide agent (MTT); ii/ DNA content by flow cytometry analysis; iii/ apoptosis determination by Annexin V and activity of caspase-3 and -7 quantification by cleavage of fluorogenic substrate CellEvent™ Caspase-3/7 Green Detection Reagent; iii/ CDKN2A and CDKN2B methylation status by methylation-specific PCR and pyrosequencing; iiii/ CDKN2A and CDKN2B gene expression analysis by quantitative RT-PCR.Results: To determine the influence of DSF, DAC and two cyclin dependent kinase (CDK) inhibitors JNJ-7706621 and SAHA on cell cycle regulation, RPMI8226 and U266 cell lines were treated with the same scheme as for methylation and expression analysis. U266 cell treatments with 1 µM DSF (18, 9 %) and 5 µM JNJ (21, 6 %) show similar parameters in sub-G1 cell population, but lower when they compared to SAHA treatment (29, 7 %). Analysis of apoptosis of cells treated with 1 µM DSF show 18,51 % of apoptotic cells, while 26,22 % and 28,4 % apoptotic cells after 5 µM SAHA and 5 µM JNJ treatments were detected. The proportions of cells in apoptosis in both 0, 1 µM and 1 µM DSF treatments are 21,8 % and 18,5 %. We have found unmethylated 162 bp CDKN2B analysed gene region, while 150 bp analysed CDKN2A gene region is methylated. Unmethylated state of the CDKN2B gene was confirmed by pyrosequencing of 223 bp promoter and 138 bp 1.exon gene regions. Expression of the unmethylated CDKN2B gene is not influenced, while the 0,1 µM DSF treatment causes increased expression of the CDKN2A gene (Graph1).Conclusion: In treated myeloma cells, we have examined apoptotic effect of DSF in comparison to CDK inhibitors JNJ-06621 and SAHA through detection of methylation and expression status of CDKN2A and CDKN2B genes. Our results indicate DSF pro-apoptotic effect comparable to used CDK inhibitors. The 5-aza-2´-deoxycytidine with demethylation activity and DSF do not affect an expression of the unmethylated CDKN2B gene, its expression effectively increases CDK inhibitors (Graph2). On the other hand, the expression of the methylated CDKN2A gene is only slightly increased in DSF treated cells in comparison to other treatments.Thisstudy was supported in part by NV18-03-0050 from the Ministry of Health of the Czech Republic and LF_2018_001 from Palacky University Olomouc. DisclosuresNo relevant conflicts of interest to declare.

The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma

ABSTRACT Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

Importance of TFEB acetylation in control of its transcriptional activity and lysosomal function in response to histone deacetylase inhibitors

ABSTRACTTFEB (transcription factor EB) is a master regulator of lysosomal biogenesis, function and autophagy. The transcriptional activity of TFEB is mainly controlled by its phosphorylation status mediated by the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) complex 1 (MTORC1). At present, little is known whether other forms of posttranslational modifications (PTMs) such as acetylation also affects is transcriptional activity. In this study, we first observed that a well-established histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) activated lysosomal function in human cancer cells, a process independent of the MTORC1 pathway. Second, SAHA treatment activated TFEB transcriptional activity, as evidenced by increased TFEB luciferase activity and expression of its target genes. Third and more importantly, we observed the enhanced TFEB acetylation in SAHA-treated cells, with identification of 4 acetylation sites. Mutation of these 4 sites markedly diminished TFEB transcriptional activity and lysosomal function induced by SAHA. Finally, we found that TFEB acetylation was functionally implicated in SAHA-mediated autophagy and cell death in cancer cells. Taken together, our results demonstrate that TFEB acetylation is a novel form of PTMs in TFEB that plays an important role in determining its transcriptional activity, lysosomal function and autophagy in cancer cells.Abbreviations: ACAT1: acetyl-coenzyme A acetyltransferase 1; AHA: L-azidohomoalanine; AO: acidic orange; ATG: autophagy related; CLEAR: Coordinated Lysosomal Expression and Regulation; CQ: chloroquine; CTSB: cathepsin B; HATs: histone acetyltransferases; HDACIs: HDACs inhibitors; HDACs: histone deacetylases; IP: immunoprecipitation; MEFs: mouse embryonic fibroblasts; MS: mass spectrometry; MTOR: mechanistic target of rapamycin (serine/threonine kinase); MTORC1: mechanistic target of rapamycin (serine/threonine kinase) complex 1; PTMs: posttranslational modifications; SAHA: suberoylanilidehydroxamic acid; TFEB: transcription factor EB

The Effect of the Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid and Paclitaxel Treatment on Full-Thickness Wound Healing in Mice.

Neoadjuvant chemotherapy prior to lumpectomy or mastectomy for breast cancer challenges wound healing. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to work synergistically with paclitaxel in vitro and in preclinical studies. In addition, our laboratory has demonstrated that SAHA treatment decreases paclitaxel-associated stem cell toxicity, modulates inflammatory response, and promotes wound healing in injured fibroblast cells. Our goal was to determine if combined SAHA and paclitaxel treatment would improve wound healing in an in vivo full-thickness murine model, without altering antitumor effect. Thirty-two nude athymic mice received intraperitoneal injections of paclitaxel (20 mg/kg), SAHA (25 mg/kg), paclitaxel + SAHA (20 mg/kg + 25 mg/kg), or no treatment for 2 weeks prior to surgery. Under general anesthesia, 8-mm full-thickness dorsal wounds were created in all animals, and a silicone splint was attached to minimize wound contraction. The wounds were measured twice a week with a surgical caliper until healing was complete. To evaluate the in vivo effect of drug treatment, 16 athymic nude mice with MDA-MB-231 xenografts received the treatments described previously, following which tumor volumes were compared between groups. Average wound healing time was prolonged in mice treated with paclitaxel (20 ± 1.9 days), and combination SAHA + paclitaxel therapy improved average wound healing time (17.0 ± 1.8 days). In the xenograft model, the antitumor effect of SAHA and paclitaxel (average tumor volume 43.9 ± 34.1 mm) was greater than paclitaxel alone (105.8 ± 73.8 mm). The addition of SAHA to taxane chemotherapy improves the therapeutic effect on triple-negative breast cancer while decreasing the detrimental effect of paclitaxel on wound healing. This may have substantial implications on improving outcomes in breast reconstruction following chemotherapy.

Alterations in histone acetylation following exposure to 60Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells.

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0-2Gy 60Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic + r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0-1Gy 60Co γ-rays. SAHA treatment significantly decreased dic + r/cell and MN/cell in cells exposed to 0.5 or 1.0Gy 60Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by 60Co γ-radiation.

Inhibitory Effect of Histone Deacetylase Inhibitor SAHA on Proliferation of Mouse Multiple Myeloma Cell Line SP2/0 in vitro and in vivo

To explore the anti-myeloma effect of suberoylanilide hydroxamic acid (SAHA) and on mouse myeloma cell line SP2/0 in vitro and in vivo and its mechanism. The inhibitory effect of SAHA on SP2/0 cells was measured by CCK-8 assay,and the apoptosis and cell cycle were analyzed by flow cytometry FACS. The protein expression of Caspase-3 and p53 of SP2/0 cells treated with SAHA were examined by Western blot. Annexin V/7-AAD double staining was performed to detect the apoptosis of SP2/0 induced by SAHA in vitro. SP2/0 cells (1×106) resuspended in 200 µl PBS were inoculated subcutaneously and intravenously into BALB/c mice, so as to establish aggressive or non-aggressive myeloma-bearing mouse models respectively. On day 3 after modeling, mice received SAHA or vehicle control treatment by intraperitoneal injection. The dose of SAHA was 60 mg/kg·d, 5 times a week for 3 weeks. In SAHA-treated SP2/0 cells, the proliferation inhibition rate and apoptotic cells increased in a dose dependent manner. Also, SAHA significantly increased the ratio of cells in G2 phase and decreased in S phase. Molecular mechanisms of apoptosis and cell cycle arrest of SP2/0 induced by SAHA partly correlated with up-regulating the expression level of Caspase-3 and p53. In the non-aggressive myeloma-bearing mice, SP2/0 cells disappeared in peripheral blood after SAHA treatment. In the aggressive myeloma-bearing mice, inhibition of tumor growth and prolongation of the cell survival were observed after SAHA treatment. SAHA inhibited SP2/0 cell proliferation, this effect associates with inducing apoptosis and cell cycle arrest, the mechanism of SAHA ralates partly with activating Caspase-3 and p53 pathway.

Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 μM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 μM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer) irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 μM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A) focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells.

The HDAC Inhibitor, SAHA, Prevents Colonic Inflammation by Suppressing Pro-inflammatory Cytokines and Chemokines in DSS-induced Colitis.

Inflammatory bowel disease (IBD) is an inflammatory disorder of the gastrointestinal tract that is caused by multiple factors, including dysfunction of the immune system and genetic and epigenetic alterations. Aberrant epigenetic regulation, especially histone acetylation, was found in biopsies from IBD patients and mouse models of colitis, suggesting that an epigenetic treatment approach may be useful for IBD therapy. Therefore, we investigated the effects of the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), in a mouse model of dextran sulfate sodium (DSS)-induced colitis. C57BL/6 mice were treated with 1.5% DSS for 5 days and/or SAHA (25 mg/kg BW/day) for 26 days. Levels of mRNA for the pro-inflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and the chemokines, Ccl2, were examined by qRT-PCR. CD11b, a marker of dendritic cells, macrophages, and monocytes, as well as Ccl2 expression, were examined by immunohistochemistry. IL-6, TNF-α, and Ccl2 gene expression peaked on day 5 in DSS-treated mouse colon, whereas SAHA treatment significantly decreased pro-inflammatory gene expression. Ccl2 protein expression resembled Ccl2 gene expression results. Moreover, localization of CD11b showed that migratory inflammatory cells were dramatically decreased by SAHA treatment compared to DSS-treated mouse colon. Thus, we conclude that the HDAC inhibitor, SAHA, attenuates inflammatory changes in DSS-induced colitis by suppressing local secretion of pro-inflammatory cytokines and chemokines and also by suppressing mobilization and accumulation of inflammatory cells.

Histone deacetylase inhibitor (HDACi) upregulates activin A and activates the Smad signaling pathway in melanomas

BackgroundHistone deacetylase (HDAC) is an enzyme that regulates gene expression, cell cycle arrest, apoptosis and modulation of various pathways. HDAC inhibitors (HDACis) can modulate these pathways by hyper-acetylating target proteins, thereby acting as cancer chemotherapeutic agents. ObjectiveOne of HDACis, suberoylanilide hydroxamic acid (SAHA), has been found to regulate the Smad signaling pathway, by an as yet unclear mechanism. This study therefore investigated the mechanism by which SAHA regulates Smad signaling in the melanoma cell line SK-Mel-5. MethodsCell proliferation was assessed by MTT assays and fluorescence activated cell sorter (FACS) analysis. The activation of Smad signaling pathway was assessed by western blots analysis. The transcriptions of target genes were checked by RT-PCR and dual luciferase assay. ResultsTreatment with SAHA inhibited the proliferation of SK-Mel-5 cells, enhanced the phosphorylation of R-Smad, and up-regulated p21 protein. Surprisingly, R-Smad was also activated by conditioned medium from SAHA-treated SK-Mel-5 cells. An analysis of the conditioned medium showed that activin A was responsible for the activation of R-Smad. SAHA treatment enhanced the level of activin A mRNA, increasing the level of activin A in the secretome. The activity of the SAHA-treated secretome could be eliminated by pre-incubation with antibody to activin A. In addition, activin A supplemented medium could mimic the effect of the SAHA-treated secretome. ConclusionThese findings indicate that the anti-cancer function of SAHA is mediated, at least in part, by the upregulation of activin A.

Histone Deacetylase (HDAC) Inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), Induces Apoptosis in Prostate Cancer Cell Lines via the Akt/FOXO3a Signaling Pathway.

BackgroundHistone deacetylase (HDAC) inhibitors are emerging as a new class of anti-cancer drugs that promote cancer cell apoptosis, and include suberoylanilide hydroxamic acid (SAHA). The aim of this study was to investigate the mechanism of SAHA-induced apoptosis in human prostate cancer cell lines, DU145 and PC-3.Material/MethodsCell lines, DU145 and PC-3, were studied before and after treatment with SAHA. The effects of SAHA treatment on cell proliferation were studied using the MTT cell proliferation assay. Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining were used to study the effects of SAHA treatment on cell apoptosis. Western blotting, quantitative polymerase chain reaction (qPCR) and short interfering (si)RNA assays were performed to study the effects of SAHA treatment on apoptotic and cell cycle proteins and the Akt/FOXO3a signaling pathway.ResultsTreatment with SAHA inhibited cell proliferation in human prostate cancer cell lines DU145 and PC-3 cells in a dose-dependent way. Cell cycle analysis and Annexin-V FITC/PI staining showed that treatment with SAHA resulted in G2/M cell cycle arrest and increased cell apoptosis in a dose-dependent way. Also, treatment with SAHA reduced the protein expression levels cyclin B and cyclin A2 and promoted the activation of FOXO3a by inhibiting Akt activation. Western blotting, the siRNA assay, and qPCR showed that FOXO3a, the Bcl-2 family of proteins, survivin, and FasL were involved in SAHA-induced apoptosis in prostate cancer cells grown in vitro.ConclusionsTreatment with SAHA promoted apoptosis via the Akt/FOXO3a signaling pathway in prostate cancer cells in vitro.

Involvement of IL-17 in Secondary Brain Injury After a Traumatic Brain Injury in Rats.

The pro-inflammatory activity of interleukin 17, which is produced by the IL-23/IL-17 axis, has been associated with the pathogenesis of traumatic brain injury (TBI). The study investigated the potential role of IL-17 in secondary brain injury of TBI in a rat model. Our data showed that the levels of IL-17 increased from 6h to 7days and peaked at 3days, in both the CNS and serum, which were consistent with the severity of secondary brain injury. The IL-23 inhibitor suberoylanilide hydroxamic acid (SAHA) treatment markedly decreased the expressions of IL-17 and apoptosis-associated proteins cleaved caspase-3 and increased the protein ratio of Bcl-2 (B cell lymphoma/leukemia-2)/Bax (Bcl-2-associated X protein). Meanwhile, neuronal apoptosis was reduced, and neural function was improved after SAHA treatment. This study suggests that IL-17 is involved in secondary brain injury after TBI. Administering an IL-23 inhibitor and thereby blocking the IL-23/IL-17 axis may be beneficial in the treatment of TBI.

Kinome expression profiling of human neuroblastoma tumors identifies potential drug targets for ultra high-risk patients

Neuroblastoma (NBL) accounts for >7% of malignancies in patients younger than 15 years. Low- and intermediate-risk patients exhibit excellent or good prognosis after treatment, whereas for high-risk (HR) patients, the estimated 5-year survival rates is still <40%. The ability to stratify HR patients that will not respond to standard treatment strategies is critical for informed treatment decisions. In this study, we have generated a specific kinome gene signature, named Kinome-27, which is able to identify a subset of HR-NBL tumors, named ultra-HR NBL, with highly aggressive clinical behavior that not adequately respond to standard treatments. We have demonstrated that NBL cell lines expressing the same kinome signature of ultra-HR tumors (ultra-HR-like cell lines) may be selectively targeted by the use of two drugs [suberoylanilide hydroxamic acid (SAHA) and Radicicol], and that the synergic combination of these drugs is able to block the ultra-HR-like cells in G2/M phase of cell cycle. The use of our signature in clinical practice will allow identifying patients with negative outcome, which would benefit from new and more personalized treatments. Preclinical in vivo studies are needed to consolidate the SAHA and Radicicol treatment in ultra-HR NBL patients.

Abstract 5844: Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, improved radiosensitivity of human hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Radiotherapy (RT) is one of modalities for HCC therapy in clinics, but the therapeutic effect is unsatisfied due to high radiosensitivity of normal liver tissues that limits the optimal dose. This study is to develop a new theraprutic approach to enhance the efficiency of RT on HCC tumors. Suberoylanilide hydroxamic acid (SAHA), a FDA approved chemotherapeutic drug, is an inhibitor of histone deacetylase (HDACs) and has been reported to enhance radiosensitivity in many cell lines. This study hypothesized that combination of SAHA and RT could improve tumor control on HCC tumors. HCC-834 cell, a primary culture of human HCC, had a dose-dependent response to RT when analyzed by clonogenic assay. SAHA had mild cytotocixity to HCC-834 cell (IC50=1μM). Combination of SAHA and RT reached a synergestic effect to prevent colony formation. In detailed, SAHA treatment did not induce DNA damage on HCC-834 cells when assayed by the expression of γH2AX. RT of 2Gy induced highest γH2AX expression within 10min and quickly returned to baseline after 24hrs. The expression of γH2AX was sustained at 24hrs when treated by SAHA and RT, and gradually recovered after 48hrs. This data indicated that SAHA inhibited the DNA repair process in HCC-834 cell lines after RT. An orthotopic tumor model was developed for further exploring the role of SAHA on HCC tumor radiotherapy. Mice were orally fed with SAHA (100mg/kg) 3hrs and 24hrs before RT of 25Gy. Mice were sacrificed at day10 after RT and maximum tumor area was examined by H&amp;E staining. Either RT (38.81 ± 15.6mm2) or SAHA alone (45.09 ± 4.493mm2) inhibited tumor growth significantly compared with control tumors (99.1 ± 0.732mm2). Combined SAHA with RT had an additive effect on tumor control (28.03±12.25mm2). These results supported our hypothsis and demonstrated that SAHA combined with local RT was effective on inhibiting DNA repair and anti-tumor growth. This study provided a new strategy on HCC tumor therapy that SAHA could act as a radiosensitizer to improve the therapeutic efficiency of RT on HCC tumors. Citation Format: Fang-Hsin Chen, Ching-Fang Yu, Ji-Hong Hong. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, improved radiosensitivity of human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5844. doi:10.1158/1538-7445.AM2017-5844

Cell-specific expression of aquaporin-5 (Aqp5) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.

BCL11B-Mediated Epigenetic Repression Is a Crucial Target for Histone Deacetylase Inhibitors in Cutaneous T-Cell Lymphoma

The treatment options for advanced cutaneous T-cell lymphoma (CTCL) are limited because of its unclear pathogenesis. Histone deacetylase (HDAC) inhibitors (HDACis) are recently developed therapeutics approved for refractory CTCL. However, the response rate is relatively low and unpredictable. Previously, we discovered that BCL11B, a key T-cell development regulator, was aberrantly overexpressed in mycosis fungoides, the most common CTCL, as compared with benign inflammatory skin. In this study, we identified a positive correlation between BCL11B expression and sensitivity to HDACi in CTCL lines. BCL11B suppression in BCL11B-high cells induced cell apoptosis by de-repressing apoptotic pathways and showed synergistic effects with suberoylanilide hydroxamic acid (SAHA), a pan-HDACi. Next, we identified the physical interaction and shared downstream genes between BCL11B and HDAC1/2 in CTCL lines. This interaction was essential in the anti-apoptosis effect of BCL11B, and the synergism between BCL11B suppression and HDACi treatment. Further, in clinical samples from 46 mycosis fungoides patients, BCL11B showed increased but varied expression in advanced tumor stage. Analysis of four patients receiving SAHA treatment suggested a positive correlation between BCL11B expression and favorable response to SAHA treatment. In conclusion, BCL11B may serve as a therapeutic target and a useful marker for improving HDACi efficacy in advanced CTCL.

Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth.

Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia‐induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI‐treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia‐induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Alleviates Salinity Stress in Cassava.

Cassava (Manihot esculenta Crantz) demand has been rising because of its various applications. High salinity stress is a major environmental factor that interferes with normal plant growth and limits crop productivity. As well as genetic engineering to enhance stress tolerance, the use of small molecules is considered as an alternative methodology to modify plants with desired traits. The effectiveness of histone deacetylase (HDAC) inhibitors for increasing tolerance to salinity stress has recently been reported. Here we use the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), to enhance tolerance to high salinity in cassava. Immunoblotting analysis reveals that SAHA treatment induces strong hyper-acetylation of histones H3 and H4 in roots, suggesting that SAHA functions as the HDAC inhibitor in cassava. Consistent with increased tolerance to salt stress under SAHA treatment, reduced Na+ content and increased K+/Na+ ratio were detected in SAHA-treated plants. Transcriptome analysis to discover mechanisms underlying salinity stress tolerance mediated through SAHA treatment reveals that SAHA enhances the expression of 421 genes in roots under normal condition, and 745 genes at 2 h and 268 genes at 24 h under both SAHA and NaCl treatment. The mRNA expression of genes, involved in phytohormone [abscisic acid (ABA), jasmonic acid (JA), ethylene, and gibberellin] biosynthesis pathways, is up-regulated after high salinity treatment in SAHA-pretreated roots. Among them, an allene oxide cyclase (MeAOC4) involved in a crucial step of JA biosynthesis is strongly up-regulated by SAHA treatment under salinity stress conditions, implying that JA pathway might contribute to increasing salinity tolerance by SAHA treatment. Our results suggest that epigenetic manipulation might enhance tolerance to high salinity stress in cassava.