Effects Of Suberoylanilide Hydroxamic Acid Research Articles

Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage.

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

Effects of Suberoylanilide Hydroxamic Acid (SAHA) on the Inflammatory Response in Lipopolysaccharide-Induced N9 Microglial Cells.

Epigenetics has shown promising results for understanding the different behaviors of microglia under the context of neuroinflammation. However, to our knowledge, the results of this complex mechanism with novel pharmacological agents such as histone deacetylase inhibitors (HDACis) are still missing. In this study, we aimed to investigate the effects of suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, on the lipopolysaccharide (LPS)-induced neuroinflammation model in the N9 microglial cells. Microglial cells were treated with SAHA (0.25, 0.5, 1.0, 1.25, 1.5 µM) and LPS (100 ng/mL) for 24 hours. Then, levels of the pro/anti-inflammatory cytokines interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), and IL-10were determined by the enzyme-linked immunosorbent assay. The total cellular HDACactivity was determined by colorimetric analysis. Additionally, the expression levels of nuclear factor kappa-B (NF-κB) were quantified via western blotting. SAHA (1.0 and 1.25 µM) attenuated theLPS-induced inflammatory response of microglial cells via decreasing NF-κB expression and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in the N9 microglial cells. Moreover, SAHA treatment improved IL-10 levels and prevented the LPS-induced increase in the HDACactivity in the microglial cells. Our results suggest SAHA attenuates the LPS-induced inflammatory response in the N9 microglial cells, and regulation of histone acetylation withHDACis might be a rational approach for the treatment of neuroinflammation.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.

Effect of suberoylanilide hydroxamic acid on peripheral blood mononuclear cell cytotoxicity towards tumor cells in canines.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACi) that suppresses the growth of tumor cells in humans and canines. SAHA reportedly enhances the antitumor activity of human peripheral blood mononuclear cell (PBMC). However, it is unclear whether a similar effect is exerted in canines. The present study focused on the effect of SAHA on the cytotoxicity of IL-2 activated PBMC in three tumor cell lines (CTAC, CIPm, and MCM-N1). The mRNA expression of a ligand for the NKG2D receptor was upregulated in SAHA-treated cell lines. Moreover, the SAHA-treated cell lines, except MCM-N1 demonstrated a significantly higher PBMC cytotoxicity compared to the untreated cell lines. Therefore, the NKG2DL upregulation likely enhanced the interaction of NKG2D-NKG2DL, leading to enhanced cytotoxicity of PBMC. It was also revealed that activated PBMC treated with SAHA significantly attenuated their cytotoxicity toward all the cell lines. Although the NKG2D, NKp46, NKp44, and NKp30 receptors, involved in PBMC cytotoxicity, were presumed to be downregulated, there was no significant reduction in the mRNA expression of these receptors. This study revealed that SAHA not only sensitizes the canine tumor cells to cytotoxicity due to PBMC activation, but also suppresses the cytotoxicity of PBMC themselves. Therefore, our results highlight the necessity of avoiding this inhibitory action to enhance the antitumor effect of SAHA in canines.

Suberoylanilide hydroxamic acid inhibits LX2 cells proliferation via decreasing yes-associated protein/transcriptional coactivator with PDZ-binding motif proteins

Abstract Background Hepatic fibrosis is a complex and dynamic process similar to “wound healing” that results in the progressive accumulation of connective tissue. We aimed to investigate the epigenetic control of liver fibrosis and Hippo pathway in human hepatic stellate cell (HSC) line. We examined the effect of Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor on the LX2 cell line. Material and methods 2.5 μM SAHA was treated to LX2 cell line for 2 days. Cell proliferation and apoptosis measurement were performed by Muse Cell Analyzer. Yes-Associated Protein/Transcrıptional Coactivator With Pdz-Binding Motif (YAP/TAZ) and alpha-smooth muscle actin (α-SMA) protein expression levels were measured by western blotting. Results In our study, we observed that the SAHA treatment reduced cell viability and induced apoptosis of LX2 cells statistically. We found that SAHA treatment decreased α-SMA, YAP and TAZ proteins levels statistically. Conclusion Decreased cell viability could be due to physiological, autophagical and also related to the apoptotical mechanisms. We thought that SAHA plays an important role in the creation of the fates of the LX2 cell line.

In vivo inhibitory effect of suberoylanilide hydroxamic acid combined with sorafenib on human hepatocellular carcinoma cells

The present study aimed to investigate the in vivo inhibitory effect of histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) combined with sorafenib on human hepatocellular carcinoma HCCLM3 cells. The nude mice transplanted with HCCLM3 cells were randomly divided into control, SAHA, sorafenib and SAHA+sorafenib groups. The nude mice in the later 3 groups were intragastrically administrated with SAHA (10 mg·kg-1·day-1), sorafenib (10 mg·kg-1·day-1) and SAHA (10 mg·kg-1·day-1) combined with sorafenib (10 mg·kg-1·day-1), respectively, for successive 20 days. Finally, the inhibition rate of tumor was measured. The expressions of MEK1/2, p-ERK1/2, Cyclin D1, Bcl-2, Bax, p53, MMP-2, MMP-9 and uPA in tumor tissues were determined. Results showed that, compared with SAHA and Sorafenib groups, in SAHA+sorafenib groups the inhibition rate of tumor was significantly increased (P < 0.05), the expression levels of MEK1/2, p-ERK1/2, Cyclin D1, Bcl-2, MMP-2 and MMP-9 and uPA protein in tumor tissues were significantly decreased, respectively (P < 0.05), and the expression levels of Bax and p53 protein were significantly increased, respectively (P < 0.05). In conclusion, compared with single drug, SAHA combined with sorafenib can enhance the inhibitory effects on HCCLM3 xenografts in nude mice.

Suberoylanilide Hydroxamic Acid Alleviates Acute Lung Injury Induced by Severe Hemorrhagic Shock and Resuscitation in Rats.

This study aimed to assess the severity of acute lung injury after mild or severe hemorrhagic shock and resuscitation, and to examine the therapeutic effects of suberoylanilide hydroxamic acid (SAHA) on lung injury. Mild and severe hemorrhagic shock were induced by total blood volume loss of 20% or 40%, respectively, which was maintained for 60 min. Then, resuscitation was performed by autologous blood and SAHA or a vehicle solution accordingly. Mean arterial pressure, heart rate, and arterial blood gas were measured during the experiment. Histological assays, wet/dry weight ratio, inflammatory cytokines, and the extent of histone acetylation were evaluated at 3 h post-resuscitation. There were no significant differences of the most indicators measured between the mild hemorrhagic shock and Sham groups. Although in severe hemorrhagic shock group, mean arterial pressure was markedly reduced, lactic acid was significantly increased after hemorrhage. Moreover, the lung injury score was increased, the wet/dry weight ratio was elevated, inflammatory factor expression levels were upregulated, the expression of phosphorylated NF-κB/p65 was enhanced, and the extent of histone acetylation was decreased at 3 h post-resuscitation. Remarkably, adjuvant treatment with SAHA decreased the lactic acid, the pathological injury score, the wet/dry weight ratio, the content of inflammatory factor, as well as the level of activated NF-κB/p65, but promoted the expression of acetylated H4. Total blood volume loss of 40% results in acute lung injury, whereas loss of 20% does not. Treatment with SAHA alleviates lung injury induced by severe hemorrhagic shock and resuscitation and the underlying mechanism involves a reversal of decreased histone acetylation and inhibition of the NF-κB pathway.

Hepatoprotective Effects of Suberoylanilide Hydroxamic Acid (A Histone Deacetylase Inhibitor) in Immunological Liver Injury in Rats

One of the main reasons for the development of chronic liver disease is autoimmune hepatitis (AIH). There is no clear etiological factor for AIH occurrence and no drug has been approved for its treatment. Vorinostat, a Histone deacetylase inhibitor (HDACi) has been shown to reduce inflammation. Our aim was to explore the therapeutic potential of vorinostat in a rat model of AIH induced by Concanavalin A (Con A). The rats were randomly divided into 4 groups; control group, con A (20 mg/kg/iv/wk) group, vorinostat (15 mg/kg/day p.o) group and Con A with vorinostat group. Blood and liver samples were collected at the end of the fourth and eighth weeks for biochemical and histopathological examinations. Results revealed that administration of Con A elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) liver enzymes, which were reduced to 52.8% and to 61.8% respectively on simultaneous administration of Vorinostat as compare to Con A group. Furthermore, Vorinostat reduced fibrosis and diminished apoptosis as deduced by the decrease in the hepatic hydroxyproline content of the liver and tumor necrosis factor-alpha levels. Relative gene expression analysis of interleukin 1b and transforming growth factor beta were significantly decreased in groups treated with vorinostat. Histopathological analysis showed that administration of vorinostat significantly restored the well organized structure of the liver, decreased initial inflammation produced by Con A, reduced liver fibrosis and mitigated hepatic stellate cell’s activities. In conclusion, administration of vorinostat in Con A model of autoimmune hepatitis, significantly decreased liver inflammation and fibrosis. So vorinostat might show a novel therapeutic approach for management of autoimmune hepatitis and its obstacles.

Synergistic Effect of Epigenetic Inhibitors Decitabine and Suberoylanilide Hydroxamic Acid on Colorectal Cancer In vitro.

Colorectal Cancer (CRC) is a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape constitute a well-documented hallmark of CRC phenotype. The accumulation of aberrant DNA methylation and histone acetylation plays a major role in altering gene activity and driving tumor onset, progression and metastasis. In this study, we evaluated the effect of Suberoylanilide Hydroxamic Acid (SAHA), a panhistone deacetylase inhibitor, and Decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco-2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco-2 cell line. On the other hand, the sequential treatment of Caco-2 cells, first with DAC and then with SAHA, induced a synergistic anti-tumor effect with a significant enhancement of growth inhibition and apoptosis induction in Caco-2 cell line as compared to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro-apoptotic proteins Bax, p53 and cytochrome c, downregulates the expression of antiapoptotic Bcl-2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti-neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β-catenin signaling. In conclusion, our results provide evidence for the profound anti-tumorigenic effect of sequentially combined SAHA and DAC in the CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Synergistic effect of epigenetic inhibitors decitabine and suberoylanilide hydroxamic acid on colorectal cancer in vitro

Colorectal cancer (CRC) is major public health burden and a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape are a well‐documented hallmark of CRC phenotype. Accumulation of aberrant DNA methylation and histone acetylation orchestrates to modify gene activity and drive tumor onset, progression and metastasis. In this study we evaluated the effect of suberoylanilide hydroxamic acid (SAHA), a pan histone deacetylase inhibitor, and decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco‐2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco‐2 cell line. On the other hand, the sequential treatment of Caco‐2 cells, first with DAC and then with SAHA, induced a synergistic anti‐tumor effect; the sequential treatment significantly enhanced growth inhibition and apoptosis of Caco‐2 cell line in comparison to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro‐apoptotic proteins Bax, p53 and cytochrome c, down regulates the expression of anti‐apoptotic Bcl‐2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti‐neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β‐catenin signaling pathways as it down regulates p‐PI3K, p‐AKT, Wnt1, p‐GSK3, cyclin A1+A2, cyclin E1 and up regulates p‐β‐catenin protein expression levels. In conclusion, our results provide evidence for the profound anti‐tumorigenic effect of sequentially combined SAHA and DAC in CRC cell line and offer new insights into the corresponding underlined molecular mechanism.Support or Funding InformationThis work was funded by SRDC, School of Arts and Sciences, Lebanese American UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

How Histone Deacetylase Inhibitors Alter the Secondary Metabolites of Botryosphaeria mamane, an Endophytic Fungus Isolated from Bixa orellana.

Fungi are talented organisms able to produce several natural products with a wide range of structural and pharmacological activities. The conventional fungal cultivation used in laboratories is too poor to mimic the natural habitats of fungi, and this can partially explain why most of the genes responsible for the production of metabolites are transcriptionally silenced. The use of Histone Deacetylase inhibitors (HDACis) to perturb fungal secondary biosynthetic machinery has proven to be an effective approach for discovering new fungal natural products. The present study relates the effects of suberoylanilide hydroxamic acid (SAHA) and sodium valproate (VS) on the metabolome of Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L. UHPLC/HR-MS analysis, integrated with four metabolomics tools: MS-DIAL, MS-FINDER, MetaboAnalyst and GNPS molecular networking, was established. This study highlighted that SAHA and VS changed metabolites in B. mamane, causing upregulation and downregulation of metabolites production. In addition, twelve compounds were detected in the extracts as metabolites structurally correlated to SAHA, indicating its important reactivity in the medium or its metabolism by the fungus. An addition of SAHA induced the production of eight metabolites while VS induced only two metabolites undetected in the control strain. This result illustrates the importance of adding HDACis to a fungal culture in order to induce metabolite production.

ING5-mediated antineuroblastoma effects of suberoylanilide hydroxamic acid.

Neuroblastoma is the most common extracranial solid neuroendocrine cancer and is one of the leading causes of death in children. To improve clinical outcomes and prognosis, discovering new promising drugs and targeted medicine is essential. We found that applying Suberoylanilide hydroxamic acid (SAHA; Vorinostat, a histone deacetylase inhibitor) and MG132 (a proteasome inhibitor) to SH‐SY5Y cells synergistically suppressed proliferation, glucose metabolism, migration, and invasion and induced apoptosis and cell cycle arrest. These effects occurred both concentration and time dependently and were associated with the effects observed with inhibitor of growth 5 (ING5) overexpression. SAHA and MG132 treatment increased the expression levels of ING5, PTEN, p53, Caspase‐3, Bax, p21, and p27 but decreased the expression levels of 14‐3‐3, MMP‐2, MMP‐9, ADFP, Nanog, c‐myc, CyclinD1, CyclinB1, and Cdc25c concentration dependently, similar to ING5. SAHA may downregulate miR‐543 and miR‐196‐b expression to enhance the translation of ING5 protein, which promotes acetylation of histones H3 and H4. All three proteins (ING5 and acetylated histones H3 and H4) were recruited to the promoters of c‐myc, Nanog, CyclinD1, p21, and p27 for complex formation, thereby regulating the mRNA expression of downstream genes. ING5 overexpression and SAHA and/or MG132 administration inhibited tumor growth in SH‐SY5Y cells by suppressing proliferation and inducing apoptosis. The expression of acetylated histones H3 and ING5 may be closely linked to the tumor size of neuroblastomas. In summary, SAHA and/or MG132 can synergistically suppress the malignant phenotypes of neuroblastoma cells through the miRNA‐ING5‐histone acetylation axis and via proteasomal degradation, respectively. Therefore, the two drugs may serve as potential treatments for neuroblastoma.

Synergistic antitumor effect of suberoylanilide hydroxamic acid and cisplatin in osteosarcoma cells.

Cisplatin, as a first-line chemotherapy drug, has been widely applied for therapy of osteosarcoma. However, its application is limited by drug resistance and serious side effects, including nephrotoxicity and ototoxicity. Suberoylanilide hydroxamic acid (SAHA) is a newly developed histone deacetylase (HDAC) inhibitor, which is the first Food and Drug Administration-approved HDAC inhibitor for the treatment of cutaneous manifestations of T-cell lymphoma. However, SAHA as a monotherapy was revealed to be limited, particularly in solid tumors. In the present study, 143B osteosarcoma cells were treated with multiple concentrations of SAHA or cisplatin, either alone or combined. The morphological characteristics of the treated cells were observed using an inverted microscope. The cytotoxicity effects of the combination of SAHA and cisplatin on 143B cells were analyzed by MTT assay, colony formation assay, wound healing cell migration assay, cell apoptosis assay and cell cycle analysis. Western blot analysis was performed to detect the protein expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2, cleaved-caspase-3, cleaved-caspase-8 and cleaved-poly (ADP-ribose) polymerase (PARP). The experimental data indicated that the inhibition of cell proliferation in the combination group was significantly increased compared with that in single drug groups. Expression levels of pro-apoptotic protein were upregulated, whereas anti-apoptotic Bcl-2 was downregulated significantly in 143B cells following SAHA/cisplatin treatment. Taken together, the results revealed that the combination of SAHA and cisplatin inhibited the proliferation of 143B cells and induced their apoptosis synergistically, and this effectiveness may be mediated by caspase activation.

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.

Effect of suberoylanilide hydroxamic acid (SAHA) on breast cancer cells within a tumor-stroma microfluidic model.

Metastatic cancer is regarded as one of the largest contributors to disease-related deaths worldwide. Poor patient prognosis and treatment outcome is tied to the lack of efficacious anti-cancer therapies, which is due in part to the lack of physiologically relevant in vitro screening systems that can mimic the native tumor microenvironment. Conventional drug-screening platforms, which are often used in the pharmaceutical industry, are either two-dimensional (2D) assays or three-dimensional (3D) hydrogel-based matrices that lack precise control over cell distribution, matrix architecture, and organization. Despite the significance of in vivo models, they have limitations as it is difficult to control and analyze the influence of specific variables within their tumor microenvironment. Thus, there is still a crucial need to develop tumor models that enable precise control of microenvironmental cues (e.g. matrix composition, soluble factors, cellular organization) to assess the efficacy of anti-cancer drugs. Herein, we report the development and validation of a 3D microfluidic invasion platform for anti-cancer drug studies. Our platform allowed for compartmentalization of tumor and stromal fibroblasts in a defined architecture, thereby enabling pharmacokinetic drug transport to a cell-dense tumor region. We analyzed the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, on the behavior of SUM159 breast cancer cells. Many HDAC inhibitors, including SAHA, have been a subject of controversy with highly conflicting results for the treatment of solid tumors in vitro as well as in clinical trials. We found that SAHA significantly inhibited cellular migration/proliferation, and decreased microtubule polarization.

Rescue of cell cycle progression in BRAFV600E inhibitor-resistant human melanoma by a chromatin modifier.

The BRAFV600E-specific inhibitor vemurafenib blocks mitogen-activated protein kinase pathway and induces cell cycle arrest at G0/G1 phase leading to apoptosis of melanomas. To gain an understanding of the dynamics of cell cycle regulation during vemurafenib therapy, we analyzed several vemurafenib-resistant human melanoma sublines derived from BRAFV600E harboring vemurafenib-sensitive parental lines. Vemurafenib provoked G0/G1 phase arrest in parental but not in vemurafenib-resistant sublines. We hypothesized that refractoriness of vemurafenib-resistant sublines to vemurafenib-mediated cell cycle inhibition can be partially rescued by the chromatin modifier suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid promoted G2/M arrest at expense of S phase irrespective of vemurafenib sensitivity. In parental lines, combination of suberoylanilide hydroxamic acid and vemurafenib induced both G0/G1 arrest and apoptosis, whereas in vemurafenib-resistant sublines combination induced G0/G1 as well as G2/M arrest resulting in dramatic cytostasis. Vemurafenib-resistant sublines exhibited extracellular signal-regulated protein kinases 1 and 2 but not AKT and hyperphosphorylation. Gene expression profiling revealed mitogen-activated protein kinase hyperactivation and deregulations of cyclins and cyclin-dependent kinases in vemurafenib-resistant sublines, all of which were reversed by suberoylanilide hydroxamic acid; changes that may explain the cytostatic effects of suberoylanilide hydroxamic acid. These results suggest that unresponsiveness of vemurafenib-resistant sublines to the biological effects of vemurafenib may be amenable by suberoylanilide hydroxamic acid. These in vitro results, while require further investigation, may provide rational biological basis for combination therapy in the management of vemurafenib-resistant melanoma.

Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells.

Objective: Laryngeal squamous cell carcinoma is one of the most common malignant tumors in the head and neck region. Due to the poor response to chemotherapeutics in patients and low survival rate, successful treatment of larynx cancer still remains a challenge. Therefore, the identification of novel treatment options is needed. We investigated the anticancer effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on two different laryngeal cancer cell lines RK33 and RK45. We also studied the antiproliferative action of SAHA in combination with cisplatin and defined the type of pharmacological interaction between these drugs. Materials and Methods: Viability and proliferation of larynx cancer cell lines were studied by methylthiazolyldiphenyl-tetrazolium bromide method and 5-bromo-2-deoxyuridine incorporation assay, respectively. The type of interaction between SAHA and cisplatin was determined by an isobolographic analysis. Western blotting, flow cytometry and quantitative polymerase chain reaction method were used to determine acetylation of histone H3, cell cycle progression and genes expression, respectively. Apoptosis was assessed by means of nucleosomes released to cytosol. Results: SAHA alone or in combination with cisplatin inhibited larynx cancer cells proliferation, whereas displayed relatively low toxicity against normal cells - primary cultures of human skin fibroblasts. The mixture of SAHA with cisplatin exerted additive and synergistic interaction in RK33 and RK45 cells, respectively. We showed that SAHA induced hyperacetylation of histone H3 K9, K14 and K23 and triggered apoptosis. SAHA also caused cell cycle arrest by upregulation of CDKN1A and downregulation of CCND1 encoding p21WAF1/CIP1 and cyclin D1 proteins, respectively. Conclusion: Our studies demonstrated that SAHA may be considered as a potential therapeutic agent against larynx tumors.

Effect of SAHA on sufentanil postconditioning-induced cardioprotection in rats with type 2 diabetes mellitus

Objective To investigate the effect of suberoylanilide hydroxamic acid(SAHA)on sufentanil postconditioning-induced cardioprotection in rats with type 2 diabetes mellitus(T2DM). Methods Male SPF Sprague-Dawley rats, weighing 250-300 g, aged 5-6 weeks, were used in the study.T2DM was induced by high-fat diet (4 weeks) and intraperitoneal 1% streptozotocin 35 mg/kg, and confirmed by fasting blood glucose level≥16.7 mmol/L.Forty rats with T2DM were divided into 5 groups(n=8 each)using a random number table: sham operation group(grou T2DM-S); ischemia-reperfusion group(group T2DM-I/R); sufentanil postconditioning group(group T2DM-SP); SAHA group(group T2DM-SA); SAHA plus sufentanil postconditioning group(group T2DM-SASP). In T2DM-SA and T2DM-SASP groups, SAHA 25 mg/kg was injected intraperitoneally once a day for 5 consecutive days before operation.Their hearts were excised and retrogradely perfused in a Langendorff apparatus.The hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion to establish the model of ischemia/reperfusion injury.At 30 min of equilibration and 30, 60 and 120 min of reperfusion, the left ventricular systolic pressure(LVSP), heart rate(HR), and the maximum rate of increase and decrease of ventricular pressure(±dp/dtmax)were recorded.At 120 min of reperfusion, the left ventricular mass(LVM)and infarct size(IS)were measured, and IS/LVM ratio was calculated.The expression of glycogen synthesis kinase 3β(GSK-3β)and phosphorylated GSK-3β(p-GSK-3β)in the myocardium was detected by Western blot. Results Compared with group T2DM-S, the LVSP, HR and ±dp/dtmax were significantly decreased, the IS and IS/LVM ratio were significantly increased, and the expression of myocardial p-GSK-3β was significantly down-regulated in group T2DM-I/R(P 0.05). Compared with group T2DM-SP, the ±dp/dtmax was significantly increased, the IS and IS/LVM ratio were significantly decreased, and the expression of myocardial p-GSK-3β was significantly up-regulated in group T2DM-SASP(P<0.05). Conclusion SAHA can improve cardioprotection induced by sufentanil postconditioning to some extent in the rats with T2DM. Key words: Hydroxamic acids; Diabetes mellitus, type 2; Sufentanil; Myocardial reperfusion injury

Transcript, methylation and molecular docking analyses of the effects of HDAC inhibitors, SAHA and Dacinostat, on SMN2 expression in fibroblasts of SMA patients

Several histone deacetylase inhibitors (HDACis) are known to increase Survival Motor Neuron 2 (SMN2) expression for the therapy of spinal muscular atrophy (SMA). We aimed to compare the effects of suberoylanilide hydroxamic acid (SAHA) and Dacinostat, a novel HDACi, on SMN2 expression and to elucidate their acetylation effects on the methylation of the SMN2. Cell-based assays using type I and type II SMA fibroblasts examined changes in transcript expressions, methylation levels and protein expressions. In silico methods analyzed the intermolecular interactions between each compound and HDAC2/HDAC7. SMN2 mRNA transcript levels and SMN protein levels showed notable increases in both cell types, except for Dacinostat exposure on type II cells. However, combined compound exposures showed less pronounced increase in SMN2 transcript and SMN protein level. Acetylation effects of SAHA and Dacinostat promoted demethylation of the SMN2 promoter. The in silico analyses revealed identical binding sites for both compounds in HDACs, which could explain the limited effects of the combined exposure. With the exception on the effect of Dacinostat in Type II cells, we have shown that SAHA and Dacinostat increased SMN2 transcript and protein levels and promoted demethylation of the SMN2 gene.

Combined Effects of Suberoylanilide Hydroxamic Acid and Cisplatin on Radiation Sensitivity and Cancer Cell Invasion in Non-Small Cell Lung Cancer.

Lung cancer is a leading cause of cancer-related mortality worldwide, and concurrent chemoradiotherapy has been explored as a therapeutic option. However, the chemotherapeutic agents cannot be administered for most patients at full doses safely with radical doses of thoracic radiation, and further optimizations of the chemotherapy regimen to be given with radiation are needed. In this study, we examined the effects of suberoylanilide hydroxamic acid (SAHA) and cisplatin on DNA damage repairs, and determined the combination effects of SAHA and cisplatin on human non-small cell lung cancer (NSCLC) cells in response to treatment of ionizing radiation (IR), and on tumor growth of lung cancer H460 xenografts receiving radiotherapy. We also investigated the potential differentiation effect of SAHA and its consequences on cancer cell invasion. Our results showed that SAHA and cisplatin compromise distinct DNA damage repair pathways, and treatment with SAHA enhanced synergistic radiosensitization effects of cisplatin in established NSCLC cell lines in a p53-independent manner, and decreased the DNA damage repair capability in cisplatin-treated primary NSCLC tumor tissues in response to IR. SAHA combined with cisplatin also significantly increased inhibitory effect of radiotherapy on tumor growth in the mouse xenograft model. In addition, SAHA can induce differentiation in stem cell-like cancer cell population, reduce tumorigenicity, and decrease invasiveness of human lung cancer cells. In conclusion, our data suggest a potential clinical impact for SAHA as a radiosensitizer and as a part of a chemoradiotherapy regimen for NSCLC. Mol Cancer Ther; 15(5); 842-53. ©2016 AACR.