In recent years, the cyclotides have emerged as the largest family of naturally cyclized proteins. Cyclotides display potent cytotoxic activity that varies with the structure of the proteins, and combined with their unique structure, they represent novel cytotoxic agents. However, their mechanism of action is yet unknown. In this work we show that disruption of cell membranes plays a crucial role in the cytotoxic effect of the cyclotide cycloviolacin O2 (1), which has been isolated from Viola odorata. Cell viability and morphology studies on the human lymphoma cell line U-937 GTB showed that cells exposed to 1 displayed disintegrated cell membranes within 5 min. Functional studies on calcein-loaded HeLa cells and on liposomes showed rapid concentration-dependent release of their respective internal contents. The present results show that cyclotides have specific membrane-disrupting activity.

The proteasome pathway is an important target for anticancer drug development. Here, we identify the antialcoholism drug disulfiram and its analogue pyrrolidine dithiocarbamate (PDTC) as inhibitors of the 26S proteasome activity in a cell-based screening assay. As expected for proteasome inhibitors, these compounds also inhibited TNF-a-induced nuclear factor-KB (NF-KB) translocation and were cytotoxic. Disulfiram was more cytotoxic against chronic lymphocytic leukemia cells compared to peripheral blood mononuclear cells (PBMC) at clinically achievable concentrations. Proteasome and NF-KB inhibition were achieved with a potency in the same range as that of the clinically used proteasome inhibitor bortezomib. Disulfiram was also able to induce accumulation of p27Kip1 and to prolong the half-life of c-Myc, both targets for proteasome-dependent degradation. It is concluded that the previously observed antitumoral and NF-KB inhibiting activity of disulfiram and PDTC could be attributed to their inhibition of the 26S proteasome. ' 2005 Wiley-Liss, Inc.Key words: disulfiram; proteasome inhibition; nuclear factor-KB; cytotoxicityThe thiocarbamate drug disulfiram has been used extensively in the treatment of alcoholism.1 Disulfiram has also been shown to induce apoptosis in a number of tumor cell lines 2-4 as well as to reduce the growth of glioma, lung carcinoma 5 and melanoma tumors in mice. 6 However, the mechanism underlying the cytotoxic and antitumoral activity of disulfiram has not been conclusively established. It has been proposed that the induction of cell death involves generation of reactive oxygen species.5 Additionally, disulfiram has been shown to inhibit nuclear factor-KB (NF-KB) in hepatoma 4 and colorectal cancer cell lines. 2 The NF-KBinhibiting activity of the disulfiram analogue pyrrolidine dithiocarbamate (PDTC) has been attributed to its antioxidative effect.7 In this work, we propose a novel mechanism for the antitumoral and NF-KB-inhibiting activities of disulfiram. Screening of a diverse pharmacological library of compounds identified disulfiram as an inhibitor of 26S proteasome activity. Our findings suggest that the antitumor effect of disulfiram could, at least partly, be attributed to its proteasome-inhibitory action, stabilizing proteasome-degraded proteins, e.g., IKB, p27Kip1 and c-Myc, subsequently resulting in the inhibition of cell-cycle progression and induction of apoptosis. Materials and methods ChemicalsCompounds used for screening were part of the LOPAC 1280 TM library (Sigma Aldrich, St. Louis, MO). The library is a collection of pharmacologically active compounds with a diverse range of proposed target receptors and enzymes. The proteasome-inhibiting substance bortezomib was provided by Millennium Pharmaceuticals (Cambridge, MA). All other chemicals were from Sigma Aldrich. Cell linesThe stably transfected human embryo kidney cell line HEK 293 expressing the ZsProSensor-1 fusion protein (HEK 293 ZsGreen, BD Biosciences, San Jose, CA) was used for screening and evaluation of prote...

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Acquired drug resistance is a major problem in cancer treatment. To explore the genes involved in chemosensitivity and resistance, 10 human tumour cell lines, including parental cells and resistant subtypes selected for resistance against doxorubicin, melphalan, teniposide and vincristine, were profiled for mRNA expression of 7400 genes using cDNA microarray technology. The drug activity of 66 cancer agents was evaluated on the cell lines, and correlations between drug activity and gene expression were calculated and ranked. Hierarchical clustering of drugs based on their drug -gene correlations yielded clusters of drugs with similar mechanism of action. Genes correlated with drug sensitivity and resistance were imported into the PathwayAssist software to identify putative molecular pathways involved. A substantial number of both proapoptotic and antiapoptotic genes such as signal transducer and activator of transcription 1, mitogen-activated protein kinase 1 and focal adhesion kinase were found to be associated to drug resistance, whereas genes linked to cell cycle control and proliferation, such as cell division cycle 25A and signal transducer of activator of transcription 5A, were associated to general drug sensitivity. The results indicate that combined information from drug activity and gene expression in a resistance-based cell line panel may provide new knowledge of the genes involved in anticancer drug resistance and become a useful tool in drug development.