Vascular disrupting activity and the mechanism of action of EHT 6706, a novel anticancer tubulin polymerization inhibitor.

Tumor blood vessels are an important emerging target for anticancer therapy. Here, we characterize the in vitro antiproliferative and antiangiogenic properties of the synthetic small molecule, 7-ethoxy-4-(3,4,5-trimethoxybenzyl)isoquinolin-8-amine dihydrochloride, EHT 6706, a novel microtubule-disrupting agent that targets the colchicine-binding site to inhibit tubulin polymerization. At low nM concentrations, EHT 6706 exhibits highly potent antiproliferative activity on more than 60 human tumor cell lines, even those described as being drug resistant. EHT 6706 also shows strong efficacy as a vascular-disrupting agent, since it prevents endothelial cell tube formation and disrupts pre-established vessels, changes the permeability of endothelial cell monolayers and inhibits endothelial cell migration. Genome-wide transcriptomic analysis of EHT 6706 effects on human endothelial cells shows that the antiangiogenic activity elicits gene deregulations of antiangiogenic pathways. These findings indicate that EHT 6706 is a promising tubulin-binding compound with potentially broad clinical antitumor efficacy.

Inhibition of RPE cell sterile inflammatory responses and endotoxin-induced uveitis by a cell-impermeable HSP90 inhibitor.

Dying cells release pro-inflammatory molecules, functioning as cytokines to trigger cell/tissue inflammation that is relevant to disease pathology. Heat-shock protein 90 (HSP90) is believed to act as a danger signal for tissue damage once released extracellularly. Potential roles of HSP90 were explored in retinal pigment epithelial (RPE) inflammatory responses to necrosis. Cellular extracts can trigger ARPE-19 cell inflammatory responses, producing cytokines that lead to an increase in ARPE-19 cell monolayer permeability. Addition of recombinant HSP90ß mimics the induction of chemokines IL-8 and MCP-1 in cultured RPE cells, suggesting that released HSP90 can incite RPE cell sterile inflammatory responses. Consistent with this, classical HSP90 inhibitors were shown to substantially reduce necrosis-induced cytokine production and permeability increases in ARPE-19 cells. Moreover, a cell-impermeable inhibitor, 17-N,N-dimethylaminoethylamino-17-demethoxy-geldanamycin-N-oxide, also efficiently inhibited necrosis-induced cytokine production and TNF-α/IL-1ß-induced increase in ARPE-19 cell permeability in vitro and endotoxin-induced development of uveitis in vivo, suggesting that HSP90 can contribute to necrosis-induced RPE inflammatory responses. Collectively, our data identify HSP90 as a pro-inflammatory molecule in RPE cell sterile inflammatory responses.

Differential effects of PPARgamma ligands on oxidative stress-induced death of retinal pigmented epithelial cells.

PURPOSE: To investigate the role of the peroxisome proliferator-activated receptor (PPAR)-γ in modulating retinal pigmented epithelium (RPE) responses to oxidative stress. METHODS: ARPE-19 cells were treated with the oxidant, t-butylhydroperoxide (tBH) to induce apoptosis. Cells pretreated with synthetic PPARγ agonists of the antidiabetic thiazolidinediones class before tBH challenge were assessed for viability and, by microarray analysis, for effects on gene expression. RESULTS: Treatment of ARPE-19 cells with tBH resulted in a loss of viability and global changes in the pattern of gene expression. PPARγ ligands were found to have differential modulatory effects on tBH-induced apoptosis of RPE cells. Whereas rosiglitazone and pioglitazone potentiated cell death, troglitazone acted as a potent cytoprotective agent. Downregulation of PPARγ expression by an siRNA resulted in enhanced cell death in response to tBH treatment and blocked the cytoprotective effect of troglitazone consistent with a role of PPARγ in mediating this response. Microarray analysis revealed that while rosiglitazone and pioglitazone had little effect on gene changes induced by tBH treatment, troglitazone dramatically reduced the number of changes caused by oxidative stress. A unique subset of genes that were deregulated by tBH and selectively normalized by troglitazone were identified. CONCLUSIONS: These findings demonstrate that PPARγ agonists can have differential effects on RPE survival in response to oxidative stress. Oxidative stress leads to deregulation of a large set of genes in ARPE-19 cells. A specific subset of these genes can be selectively modulated by troglitazone and represent potential novel targets for cytoprotective therapies.