Vorinostat (SAHA, MK0683)

Histone deacetylase inhibitor vorinostat (SAHA, MK0683) perturb miR-9-MCPIP1 axis to block IL-1β-induced IL-6 expression in human OA chondrocytes

Aim of the study: High levels of IL-6 are believed to contribute to osteoarthritis (OA) pathogenesis. The expression of IL-6 is regulated post-transcriptionally by the miR-9-MCPIP-1 axis in chondrocytes. Vorinostat (SAHA) inhibits the IL-6 expression in OA chondrocytes. We investigated whether SAHA suppresses the expression of IL-6 by perturbing the miR-9-MCPIP1 axis in OA chondrocytes under pathological conditions. Materials and methods: OA chondrocytes were isolated by enzymatic digestion and treated with IL-1β in the absence or presence of SAHA. Genes and protein expression levels were determined by TaqMan assays and Western blotting, respectively. Secreted IL-6 was quantified by enzyme linked immunosorbent assay (ELISA). MCPIP1 promoter deletion mutants were generated by polymerase chain reaction (PCR). Promoter recruitment of transcription factors was determined by ChIP. Nuclear run-on was employed to measure the ongoing transcription. siRNA-mediated knockdown of the CEBPα expression was employed for loss of function studies. Results: Expression of MCPIP1 was high in SAHA treated OA chondrocytes but expression of IL-6 mRNAs and secreted IL-6 were reduced by ~70%. SAHA suppressed the expression of miR-9 but enhanced the activity of the MCPIP1 promoter localized to a 156bp region which also harbors the binding site for CEBPα. Treatment with SAHA enhanced the recruitment of CEBPα to the MCPIP1 promoter. Ectopically expressed CEBPα enhanced the promoter activity and the expression of MCPIP1 while siRNA-mediated knockdown of CEBPα inhibited the expression of MCPIP1. Conclusions: Taken together our data indicate that SAHA-mediated suppression of the IL-6 expression is achieved through increased recruitment of CEBPα to the MCPIP1 promoter and by relieving the miR-9-mediated inhibition of MCPIP1 expression in OA chondrocytes.

Vorinostat (SAHA) May Exert Its Antidepressant-Like Effects Through the Modulation of Oxidative Stress Pathways

Suberoylanilide hydroxamic acid (SAHA/Vorinostat), a potent inhibitor of histone deacetylases (HDACs), is known to possess antidepressant properties. However, the exact mechanisms underlying this activity are unknown. In this study, we evaluated the effect of SAHA on the expression of GluN2A, GluN2B (NMDA receptor subunits), (p-)AMPK, and ΔFos proteins which are an integral part of the signal transduction pathways in the brain and also involved in the pathophysiology of depression as well as the mechanism of antidepressant action. We also measured the concentration of malondialdehyde (MDA - a product of lipid peroxidation). The study was carried out in the prefrontal cortex (PFC) and hippocampus (Hp), brain regions implicated in depression. Although SAHA induced changes in the expression of all the proteins and MDA concentration, the effects differed depending on the drug dose, time, and brain structure involved. SAHA reduced MDA concentration and significantly increased p-AMPK protein expression, indicating it may prevent oxidative stress. SAHA also increased the levels of HDAC3 and NMDA subunits (GluN2A and GluN2B), implying it is neuroprotective and may play a crucial role in synaptic plasticity. Moreover, ΔFosB and FosB levels were significantly elevated, suggesting that SAHA may modulate learning and memory processes. Overall, the data indicate that the Hp might play a pivotal role in the mechanism of action of SAHA, hinting at novel mechanisms it play in the antidepressant and neuroprotective effects of SAHA.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM.
Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays.
Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner.
Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.

Cell Cycle Arrest and Cytotoxic Effects of SAHA and RG7388 Mediated through p21WAF1/CIP1 and p27KIP1 in Cancer Cells

Background and objective: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells.
Materials and methods: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods.
Results: The RG7388 treatment was able to induce cell death by elevating p21^WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21^WAF1/CIP1 in MCF-7 cells.
Conclusion: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi.

Synergistic effects of vorinostat (SAHA) and azoles against Aspergillus species and their biofilms

Background: Invasive aspergillosis is a fungal infection that occurs mainly in immunocompromised patients. It is responsible for a high degree of mortality and is invariably unresponsive to conventional antifungal treatments. Histone deacetylase inhibitors can affect the cell cycle, apoptosis and differentiation. The histone deacetylase inhibitor vorinostat (SAHA) has recently received approval for the treatment of cutaneous T cell lymphoma. Here, we investigated the interactions of SAHA and itraconazole, voriconazole, and posaconazole against Aspergillus spp. in vitro using both planktonic cells and biofilms.
Results: We investigated 20 clinical strains using broth microdilution checkerboard methods. The results showed synergy between SAHA and itraconazole, voriconazole, and posaconazole against 60, 40, and 25% of tested isolates of planktonic Aspergillus spp., respectively. Similar synergy was also observed against Aspergillus biofilms. The expression of the azole-associated multidrug efflux pumps MDR1, MDR2, MDR3 and MDR4, as well as that of HSP90, was measured by RT-PCR. The results indicated that the molecular mechanism of the observed synergistic effects in Aspergillus fumigatus may be partly associated with dampened expression of the efflux pump genes and, furthermore, that HSP90 suppression may be a major contributor to the observed synergistic effects of the drugs.
Conclusions: SAHA has potential as a secondary treatment to enhance the effects of azoles against both biofilm and planktonic cells of Aspergillus spp. in vitro. This effect occurs mostly by inhibition of HSP90 expression.