BACKGROUND:Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase‐independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV‐128 as an inducer of cell death through a caspase‐independent pathway.METHODS:Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV‐128, and the concentration that caused 50% growth inhibition (GI50) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein‐protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model.RESULTS:NV‐128 was able to induce significant cell death in both paclitaxel‐resistant and carboplatin‐resistant EOC cells with a GI50 between 1 μg/mL and 5 μg/mL. Cell death was characterized by chromatin condensation but was caspase‐independent. The activated pathway involved the down‐regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin‐1 followed by nuclear translocation of endonuclease G.CONCLUSIONS:The authors characterized a novel compound, NV‐128, which inhibits mTOR and promotes caspase‐independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase‐dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV‐128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society.

The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-X L :BAD, BCL-X L :BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes. With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial. The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.

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A preparação de benzodiazepínicos 1,4 substituídos na posição 3 é explorada a partir da alquilação de enolatos benzodiazepínicos. Empregando-se tal abordagem, vários gramas de benzodiazepínicos 1 foram preparados para estudos em animais, visando avaliar uma nova abordagem no tratamento da doença auto-imune lúpus eritematoso (LE). The preparation of 3-substituted 1,4-benzodiazepines by benzodiazepine enolate alkylation has been explored. Employing this approach, multigram quantities of benzodiazepine 1 have been prepared for animal studies to evaluate a new approach for the treatment of the autoimmune disease systemic lupus erythematosus (SLE).