Trichostatin A (TSA) is an anticancer drug that inhibits histone deacetylases (HDACs). Hypoxia-inducible factor 1 (HIF-1) participates in tumor angiogenesis by upregulating target genes, such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether TSA treatment increases HIF-1α stabilization via acetylation under normoxic conditions, which would lead to VEGF upregulation and resistance to anticancer drugs. TSA enhanced total HIF-1α and VEGF-HRE reporter activity under normoxic conditions. When cells were transfected with GFP-HIF-1α, treatment with TSA increased the number of green fluorescence protein (GFP)-positive cells. TSA also enhanced the nuclear translocation of HIF-1α protein, as assessed by immunoblotting and as evidenced by increased nuclear localization of GFP-HIF-1α. An increase in the interaction between HIF-1α and the VEGF promoter, which was assessed by a chromatin immunoprecipitation (ChIP) assay, led to activation of the VEGF promoter. TSA acetylated HIF-1α at lysine (K) 674, which led to an increase in TSA-induced VEGF-HRE reporter activity. In addition, TSA-mediated cell death was reduced by the overexpression of HIF-1α but it was rescued by transfection with a HIF-1α mutant (K674R). These data demonstrate that HIF-1α may be stabilized and translocated into the nucleus for the activation of VEGF promoter by TSA-mediated acetylation at K674 under normoxic conditions. These findings suggest that HIF-1α acetylation may lead to resistance to anticancer therapeutics, such as HDAC inhibitors, including TSA.

Actin plays a role in various processes in eukaryotic cells, including cell growth and death. We investigated whether the antitumor effect of trichostatin A (TSA) is associated with the dynamic rearrangement of F-actin. TSA is an antitumor drug that induces hyper-acetylation of histones by inhibiting histone deacetylase. HeLa human cervical cancer cells were used to measure the antitumor effect of TSA. The percent cell survival was determined by an MTT assay. Hypodiploid cell formation was assessed by flow cytometry. Collapse of the mitochondrial membrane potential (MMP) was identified by a decrease in the percentage of cells with red MitoProbe J-aggregate (JC-1) fluorescence. Cell survival was reduced by treatment with TSA, as judged by an MTT assay and staining with propidium iodide, FITC-labeled annexin V, or 4',6-diamidino-2-phenylindole (DAPI). TSA also induced an MMP collapse, as judged by the measurement of intracellular red JC-1 fluorescence. In addition, the F-actin depolymerizers cytochalasin D (CytoD) and latrunculin B (LatB) induced an MMP collapse and increased apoptotic cell death in HeLa cells. However, our data show that apoptotic cell death and the MMP collapse induced by TSA were decreased by the co-treatment of cells with CytoD and LatB. These findings demonstrate that the dynamic rearrangement of F-actin might be necessary for TSA-induced HeLa cell apoptosis involving a TSA-induced MMP collapse. They also suggest that actin cytoskeleton dynamics play an important role in maintaining the therapeutic effects of antitumor agents in tumor cells. They further suggest that maintaining the MMP could be a novel strategy for increasing drug sensitivity in TSA-treated tumors.

Abstract Rheumatoid arthritis (RA), one of autoimmune diseases, is characterized by histological hallmarks such as hyperplasia of the synovial lining cells, mononuclear cell infiltration, angiogenesis, and osteoclastic bone destruction. Angiogenesis is mediated through expression of vascular endothelial growth factor (VEGF) in RA synovium. VEGF expression is enhanced by TNF-α, the main pro-inflammatory cytokine in RA. B cell activating factor (BAFF) which plays a role in maturation and maintenance of B cells is also associated with autoimmune RA. Here, we investigated whether BAFF could regulate VEGF expression in MH7A synovial cells that is established by transfection with the SV40 T antigen (Miyazawa et al., 1998). When MH7A cells were treated with TNF-α, data showed that TNF-α increased the expression of BAFF and VEGF as judged by western blotting, RT-PCR and luciferase promoter assay. Inhibition of BAFF expression with BAFF-siRNA decreased VEGF expression. In addition, BAFF and VEGF expression was mediated by nuclear translocation of c-Fos activated by the treatment with TNF-α as judged by the transfection of MH7A cells with c-fos-siRNA. In conclusion, our results suggest for the first time that angiogenesis in RA could be controlled by BAFF-mediated VEGF expression.