Inhibitory effect of vorinostat encapsulated hydroxypropyl-β-cyclodextrin eye drops on corneal neovascularization induced by alkali burns in mouse / 中华实验眼科杂志

Objective:To prepare vorinostat encapsulated hydroxypropyl-β-cyclodextrin (SAHA-CD) eye drops and investigate its inhibitory effect on corneal neovascularization (CNV) induced by alkali burns in mouse.Methods:The SAHA-CD eye drops at concentrations of 0.1%, 0.2%and 0.4%were prepared by inclusion technology with hydroxypropyl-β-cyclodextrin, and the content was assayed by high performance liquid chromatography.Seventy-five SPF mice with alkali burn-induced CNV were randomized into 0.1%SAHA-CD group, 0.2%SAHA-CD group, 0.4%SAHA-CD group, dexamethasone group and normal control group according to a random number table, 15 for each group, among which the SAHA-CD groups and dexamethasone group were treated with corresponding drugs, and model control group was treated with normal saline immediately after modeling, four times a day and five microliters each time, lasting for six days.The healing of corneal epithelium was examined with a slit lamp microscope after fluorescein sodium staining, and the areas of cornea epithelial defects were calculated using Eyestudio software.The corneal flat mount was prepared, and the length and areas of CNV were calculated with ImageJ software.The histology of mouse corneas was observed through hematoxylin and eosin staining.The expression level of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-9 (MMP-9) in cornea were measured with enzyme linked immunosorbent assay (ELISA) kits.The use and care of animals complied with the ARVO statement and this study protocol was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (No.HNEECA-2020-01).Results:The actual drug contents of the 0.1%, 0.2% and 0.4%SAHA-CD eye drops were 97.62%, 98.33%and 98.14%of the labeled amount.The cornea showed edema and opacification after modeling.On the sixth day after treatment, significant differences were found in the length and areas of CNV among various groups ( F=7.655, 8.802; both at P<0.01).The areas of CNV in 0.2%SAHA-CD, 0.4%SAHA-CD and dexamethasone groups were significantly smaller than model control group, and the length of CNV in 0.1%SAHA-CD, 0.2%SAHA-CD and dexamethasone groups were significantly smaller than model control group (all at P<0.05).On the third and sixth day following modeling, significant differences in the expression levels of VEGF, bFGF and MMP-9 were found among the five groups (third day: F=6.345, 7.149, 18.650; all at P<0.01; sixth day: F=6.749, 5.105, 5.023; all at P<0.01), and the expression levels of VEGF, bFGF and MMP-9 in 0.2%SAHA-CD group were significantly lower than those in 0.1%SAHA-CD group, 0.4%SAHA-CD group and model control group (all at P<0.05). Conclusions:SAHA-CD eye drops can inhibit alkali burn-induced CNV in mouse.

The mechanism of parthenolide strengthen vorinostat on inhibiting the proliferation of A549 non-small cell lung cancer cells / 药学学报

This research explored the synergistic effects and the mechanism of parthenolide (PTL) and vorinostat (suberoylanilide hydroxamic acid, SAHA) on the proliferation of A549 non-small cell lung cancer cells. The combination effect of PTL and SAHA was detected by cell counting kit-8 (CCK-8) and colony formation assays. Scratch test was performed to detect cell migration. Annexin V-fluorescein isothiocyanate isomer/propidium iodide (FITC/PI) flow cytometry and Western blot analyses were used to determine cell apoptosis and its mechanism. The results showed that combination of PTL and SAHA inhibited the proliferation and migration of A549 with a synergistic effect compared to the single-drug groups. The combination of PTL and SAHA had synergistic effect to induce cell apoptosis by regulating p53 and c-myc pathways, and affected the expression levels of poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase (caspase)-9, and caspase-3. Taken together, this study shows that combination of PTL and SAHA has synergistic effect, induces cell apoptosis and inhibits A549 proliferation, which is likely to be a novel strategy for the treatment of non-small cell lung cancer.

Epigenetic therapies in patients with solid tumors: Focus on monotherapy with deoxyribonucleic acid methyltransferase inhibitors and histone deacetylase inhibitors

Epigenomics is the study of the gene expression changes due to epigenetic processes and not due to the deoxyribonucleic acid (DNA) base sequence alterations. The key mechanisms of epigenetic regulation include DNA methylation, histone modifications, and noncoding RNAs. Epigenetic alterations in cancer are predominantly linked with hypermethylation of promoters of the tumor suppressor genes, global DNA hypomethylation, and increased expression of histone deacetylases (HDAC). There is a growing need to investigate epigenetic patterns and to provide safe and effective, innovative therapeutic strategies for oncology patients, who did not improve on traditional anticancer regimens. The epi-drugs (e.g., DNA methyltransferase inhibitors, e.g., azacitidine and decitabine and HDAC inhibitors, e.g., vorinostat and romidepsin) have been approved for the clinical use. In this paper, we provide a brief overview of the mechanisms of action and targets for novel epi-drugs, focusing on their potential clinical applications in patients with solid tumors, resistant to standard oncology treatments

Effect of auranofin combined with antineoplastic vorinostat on bactericidal activities against gramnegative bacteria and study on the target of auranofin / 中华传染病杂志

Objective To find the target of auranofin with the antibacterial activity against gramnegative bacteria and to investigate the effect of the combination of auranofin and vorinostat on the antibacterial activity against gram-negative bacteria.Methods The strains of E.coli lacking thioredoxin reductase (TrxR)was used to find the target gene.The potential synergies of the combination of auranofin and vorinostat for E.coli strain,A.baumannii strain,P.aeruginosa strain,K.pneumonia strain and muhidrug-resistant (MDR)A.baumannii strain were evaluated using susceptibility tests,micro-dilution checkerboard tests and time-kill studies.The genes related to Trx (trxA,trxB,trxC) and the gene expressed glutathione (gor) of E.coli BW25113 strains (WT) were separately knocked out to observe the effect of auranofin on minimum inhibitory concentration (MIC) and the time-kill kinetics of △trxC and △gor.Furthermore,the complemented strains (C-trxA,C-trxB,C-trxC,C-gor) were used to verify and define the genetic targets.Results According to the results of susceptibility tests,MICs of auranofin were 64 mg/L for E.coli strain BW25113 and K.pneumonia strain ATCC 43816,128 mg/L for P.aeruginosa strain PA14 and 32 mg/L for both A.baumannii strain ATCC 17978 and A.baumannii strain AB5075.However,MICs of vorinostat are 512 mg/L for all isolates.The fractional inhibitory concentration indexes (FICIs) of the combination of auranofin and vorinostat for E.coli strain BW25113,A.baumannii strain ATCC 17978,MDR A.baumannii strain AB5075,K.pneumonia starin ATCC 43816 and P.aeruginosa strain PAl4 were 0.313,0.375,0.375,0.375,and 0.375,respectively,with all values ＜ 0.5,which showed synergy.In susceptibility tests of knockout strains,MICs of auranofin for △trxC increased from 64 mg/L to 256 mg/L,decreased to 16 mg/L for △gor,and no changes for △trxA and △trxB.Auranofin showed the same antibacterial activities against the complemented strains (C-trxC,C-gor) and E.col BW25113,which decreased by about 1.8 lg colong formins units (CFU)/mL of bacterial counts.However,the antibacterial activity of auranofin was significantly reduced for △trxC,and decreased by ＜ 1 lg CFU/mL of bacterial counts.For Agor,bacterial counts decreased 4.6 lg CFU/mL,and the antibacterial activity markedly increased.Conclusions The potential target gene of auranofin against gram-negative bacteria could be trxC,which provides new ideas and methods for the clinical treatment of multidrug-resistant gram-negative bacteria.

Effects of vorinostat in combination with cisplatin on proliferation and apoptosis of human osteosarcoma cells / 肿瘤

Objective: To investigate the effects of histone deacetylase inhibitor (HDACi) vorinostat in combination with cisplatin on the proliferation, cell cycle and apoptosis of human osteosarcoma U2OS cells, and to explore the possible mechanisms. Methods: After the treatment with vorinostat and cisplatin, the proliferation of U2OS cells was detected by MTS method, while the cell cycle distribution, mitochondrial membrane potential and apoptosis of U2OS cells were detected by FCM. Furthermore, the effects of vorinostat in combination with cisplatin on the expressions of caspase-3, caspase-8, caspase-9, poly ADP-ribose polymerase (PARP), c-Jun N-terminal kinase (JNK) and phospho-JNK (p-JNK) proteins in U2OS cells were detected by Western blotting. Results: The proliferation of U2OS cells was inhibited by vorinostat in combination with cisplatin (P < 0.05), and the cell cycle was arrested at S phase (P < 0.05). The mitochondrial membrane potential of U2OS cells was decreased (P < 0.01), and the apoptosis of U2OS cells was induced by vorinostat in combination with cisplatin (P < 0.05). The expression levels of caspase-3, caspase-8, caspase-9, PARP and p-JNK proteins in U2OS cells after the treatment with vorinostat and cisplatin were down-regulated (all P < 0.05). Conclusion: Vorinostat in combination with cisplatin can induce the cell cycle arrest and apoptosis of osteosarcoma cells as well as suppress the proliferation. The mechanism may be associated with JNK/caspase signaling pathway.

The effects of vorinostat on erlotinib-resistance of lung adenocarcinoma cells / 安徽医科大学学报

Objective To investigate the impact of vorinostat(SAHA) on the erlotinib-resistance of human lung adenocarcinoma cell line PC-9/ER.Methods PC-9/ER cells were treated with erlotinib and SAHA,alone or in combination.The effects of proliferation inhibition were assayed by MTT method,the apoptosis ratios of cells were analyzed by flow cytometry.Results The results showed that SAHA inhibited the proliferation of PC-9/ER cells in a dose-dependent manner alone.The non-toxic doses of SAHA (1 μmol/L) significantly improved the sensitivity of PC-9/ER to Erlotinib,and induce apoptosis.Conclusion SAHA could partially improve PC-9/ER sensitivity to erlotinib.

Effects of SAHA on hippocampal TLR4/MYD88 signaling and neuronal apoptosis after seizure in developing rats / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of vorinostat ( suberoylanilide hydroxamic acid, SAHA) , a histone deacetylase inhibitor, on seizure-induced brain damage in developing rats and its mechanism.METHODS:Male Sprague-Dawley rats (n=32) were randomly divided into control group, pentylenetetrazole (PTZ) group, PTZ+10 mg/kg SAHA group and PTZ+50 mg/kg SAHA group.Intraperitoneal injection of PTZ was used to induce rat seizure.SAHA was injec-ted intraperitoneally 2 h before PTZ injection.The rats in different seizure stages were counted and mean seizure score was analyzed at 30~60 min after PTZ injection.Hippocampal tissues were sampled at 24 h after seizures.The expression of TLR4, MYD88, NF-κB P65 and IL-1βat mRNA and protein levels was detected by RT-qPCR and Western blot, respec-tively.The pathological changes of the brain tissues were observed by HE staining.The apoptotic neurons were observed by TUNEL staining.RESULTS:The mRNA and protein levels of TLR4, MYD88, NF-κB P65 and IL-1β, the apoptosis of neurons, the inflammation reaction and mean seizure score significantly increased after PTZ treatment (P<0.05), and these effects were attenuated by treatment with SAHA.Compared with PTZ+10 mg/kg SAHA group, PTZ+50 mg/kg SA-HA group showed more significant protective effect against seizure-induced brain damage.CONCLUSION: Histone deacetylase inhibitor SAHA suppresses seizure-induced TLR4/MYD88 signaling and reduces apoptosis of neurons, sugges-ting a protective effect against brain damage associated with seizure in developing rats.

Histone deacetylase inhibitors: pharmacotherapeutic implications as epigenetic modifier.

Epigenetic modifications such as acetylation and deacetylation of histone proteins play a decisive role in transcriptional alteration and expression of genes. Acetylation is catalysed by the histone acetyl transferases enzymes and activates expression of genes by converting chromatin into a less compact, transcriptionally active state. Histone deacetylases enzymes catalyze deacetylation that condenses chromatin into a closed structure .Consequently transcriptional factors are unable to access DNA and gene expression is suppressed. Balanced activity of HATs and HDACS is essential for normal gene expression. Increased HDAC activity can lead to imbalance in protein acetylation resulting in hypoacetylation, tight chromatin structure and suppression of various genes. This aberrant suppression of genes is the hallmark of several malignant and other diseases including neurodegenerative disorders. Histone Deacetylase Inhibitors (HDACIs) have potential to restore the balance of histone acetylation that reverses the silencing of pathological genes. Thus HDACIs modify expression of genes without affecting sequence of DNA and act as epigenetic modifiers. Vorinostat and romidepsin are FDA approved HDACIs. Valproic acid, belinostat and many others are in different phases of clinical trials. This review article explores the target based epigenetic mechanisms as well as existing and potential therapeutic role of HDACIs in various malignant and non-malignant diseases. Data sources were articles published in medical journals and bibliographic database Medline.

Transactivation of bad by vorinostat-induced acetylated p53 enhances doxorubicin-induced cytotoxicity in cervical cancer cells

Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.

Synergistic effect of vorinostat and melphalan on human multiple myeloma cell lines / 肿瘤研究与临床

Objective To observe the anti-tumor effect on human multiple myeloma cell lines U266 and KM3 with a combination of varinostat and melphalan in vitro.Methods The cell proliferation of U266 and KM3 was datected with MTT assay when treated them with vorinostat alone and melphalan alone,then calculate their IC50 values respectively.Fixed the concentrations of vorinostator melphalan,the cell proliferation was datected in combination with melphalan/vorinostat in seriesly concentrations by MTr assay.Then to calculate drug combination index(CI).Results The IC50 value of U266 was 5.0-7.5 μmol/L and that of KM3 was 2.5-5.0 μmol/L when treated by vorinostat alone,the IC50 value of U266 was 40-60 μmol/L and that of KM3 was 60-80 μmol/L treated by melphalan alone.When fixed the concentration of vorinostat(in U266 the concentration was 1.25 μmol/L,in KM3 the concentration was 1.0 μ mol/L),Synergism(CI＜0.9)was observed when the concentrations of melphalan were 20 μmol/L,40 μmol/L,60 μ mol/L and 80 μ mol/L in U266,40 μmol/L,60 μmol/L,80 μmol/L and 100 μmol/L in KM3;When fixed the concertration of melphalan (in U266,was 10 μmol/L,in KM3 was 20 μmol/L),synergism(CI＜0.9)was observed when the concentrations of vorinostat were 1.0 μmol/L,2.5 μmol/L,5.0 μmol/L and 7.5 μmol/L in U266,and 1.0 μmol/L,2.5μmol/L,5.0 μmol/L in KM3.An additive effect was observed with the czombination of vorinostat 7.5 μmol/L plus melphalan 20 μmol/L in KM3(CI=0.93).Conclusion Vorinostat had potential anti-myeloma effect alone,and had synergistic anti-tumor effect with melphalan in vitro.

Histone deacetylase inhibitor KBH-A42 inhibits cytokine production in RAW 264.7 macrophage cells and in vivo endotoxemia model

In light of the anti-inflammatory properties of histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), we examined a new HDAC inhibitor KBH-A42 for its anti-inflammatory activities. KBH-A42 showed noteworthy anti-inflammatory properties in vitro via suppression of the production of TNF-alpha, a proinflammatory cytokine, and nitric oxide (NO), a proinflammatory effector molecule, in LPS-stimulated RAW264.7 cells and peritoneal macrophages. It also inhibited TNF-alpha production in vivo as demonstrated in a LPS-induced mouse endotoxemia model. The levels of TNF-alpha, IL-1beta, IL-6 and iNOS mRNAs determined by RT-PCR propose that the inhibition of these pro-inflammatory mediators by KBH-A42 resulted from inhibiting expression of these genes. However, the EMSA study to see the effect of KBH-A42 on the binding of NF-kappaB, a transcription factor, to a specific DNA sequence showed that the binding of NF-kappaB to DNA was not changed regardless of increasing the concentration of KBH-A42 in the presence and absence of LPS stimulation. Interestingly, DNA binding of another transcription factor AP-1 dose-dependently increased by KBH-A42. KBH-A42 differentially regulated the phosphorylation of MAP kinases. While the phosphprylation of ERK1/2 and SAPK/JNK was not affected by KBH-A42, the phosphorylation of p38 decreased by KBH-A42. These results showed that KBH-A42 inhibits production of proinflammatory cytokines in macrophages by decreasing their mRNA levels, and p38 kinase is involved in the KBH-A42-mediated inhibition.