Abnormal vascularization of solid tumours results in the development of microenvironments deprived of oxygen and nutrients that harbour slowly growing and metabolically stressed cells. Such cells display enhanced resistance to standard chemotherapeutic agents and repopulate tumours after therapy. Here we identify the small molecule VLX600 as a drug that is preferentially active against quiescent cells in colon cancer 3-D microtissues. The anticancer activity is associated with reduced mitochondrial respiration, leading to bioenergetic catastrophe and tumour cell death. VLX600 shows enhanced cytotoxic activity under conditions of nutrient starvation. Importantly, VLX600 displays tumour growth inhibition in vivo. Our findings suggest that tumour cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, and suggest a strategy for targeting the quiescent populations of tumour cells for improved cancer treatment.

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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Cardiac glycosides have been reported to exhibit cytotoxic activity against several different cancer types, but studies against colorectal cancer are lacking. In a screening procedure aimed at identifying natural products with activity against colon cancer, several cardiac glycosides were shown to be of interest, and five of these were further evaluated in different colorectal cancer cell lines and primary cells from patients. Convallatoxin (1), oleandrin (4), and proscillaridin A (5) were identified as the most potent compounds (submicromolar IC50 values), and digitoxin (2) and digoxin (3), which are used in cardiac disease, exhibited somewhat lower activity (IC50 values 0.27-4.1 microM). Selected cardiac glycosides were tested in combination with four clinically relevant cytotoxic drugs (5-fluorouracil, oxaliplatin, cisplatin, irinotecan). The combination of 2 and oxaliplatin exhibited synergism including the otherwise highly drug-resistant HT29 cell line. A ChemGPS-NP application comparing modes of action of anticancer drugs identified cardiac glycosides as a separate cluster. These findings demonstrate that such substances may exhibit significant activity against colorectal cancer cell lines, by mechanisms disparate from currently used anticancer drugs, but at concentrations generally considered not achievable in patient plasma.

The myeloma cell line RPMI 8226/S and its doxorubicin resistant subline 8226/Dox40 were used as models to explore the potential importance of the STAT1 signaling pathway in drug and radiation resistance. The 40-fold doxorubicin resistant subline 8226/Dox40 was found to be crossresistant to single doses of 4 and 8 Gy of radiation. A genome-wide mRNA expression study comparing the 8226/Dox40 cell line to its parental line was performed to identify the underlying molecular mechanisms. Seventeen of the top 50 overexpressed genes have previously been implicated in the STAT1 signaling pathway. STAT1 was over expressed both at the mRNA and protein level. Moreover, analyses of nuclear extracts showed higher abundance of phosphorylated STAT1 (Tyr 701) in the resistant subline. Preexposure of the crossresistant cells to the STAT1 inhibiting drug fludarabine reduced expression of overexpressed genes and enhanced the effects of both doxorubicin and radiation. These results show that resistance to doxorubicin and radiation is associated with increased STAT1 signaling and can be modulated by fludarabine. The data support further development of therapies combining fludarabine and radiation. ' 2006 Wiley-Liss, Inc.Key words: drug resistance; radiation resistance; STAT1; microarray Failure of cancer treatment with chemo-as well as radiotherapy is often caused by resistance at the cellular level. The molecular mechanisms underlying drug resistance have to a large extent been elucidated through experiments in vitro, with cell lines selected for resistance. The mechanisms include efflux pumps, glutathionebased detoxification systems and DNA repair.1 The primary mechanism for killing cells by ionizing radiation is through DNA damage. Thus, the search for molecular mechanisms that can explain induced radio resistance has largely focused on DNA repair. 2DNA repair systems such as nucleotide excision repair and repair of double strand breaks have been implicated in resistance to both radiation and drugs. [3][4][5] Signaling through the signal transducer and activator of transcription 1 (STAT1) has recently been suggested as an important mechanism for resistance to radiation.6,7 Furthermore, we recently demonstrated that STAT1 expression correlates positively with doxorubicin resistance in a human tumor cell line panel.8 STAT1 signaling has also been shown to mediate resistance to the platinum drug AMD473 in ovary cancer cells. 9 Understanding the underlying molecular mechanisms of cellular crossresistance is of particular interest, since it may open for therapeutic strategies to counteract cellular crossresistance and thus improve treatment.In this work we used expression profiling and a pharmacological approach to explore the hypothesis that STAT1 signaling is one mechanism that contributes to acquired crossresistance to both drugs and radiation in myeloma cell lines. Material and methods Cell cultureThe cell line RPMI 8226/S and its subline RPMI 8226/Dox40 (kind gifts from WS Dalton, AZ) were grown in culture medium RPMI-1640 supple...

The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor ␤ signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery. Molecular & Cellular Proteomics 9:178 -183, 2010.

A high-throughput screen of the cytotoxic activity of 2000 molecules from a commercial library in three human colon cancer cell lines and two normal cell types identified the acridine acriflavin to be a colorectal cancer (CRC) active drug. Acriflavine was active in cell spheroids, indicating good drug penetration and activity against hypoxic cells. In a validation step based on primary cultures of patient tumor cells, acriflavine was found to be more active against CRC than ovarian cancer and chronic lymphocytic leukemia. This contrasted to the activity pattern of the CRC active standard drugs 5-fluorouracil, irinotecan and oxaliplatin. Mechanistic studies indicated acriflavine to be a dual topoisomerase I and II inhibitor. In conclusion, the strategy used seems promising for identification of new diagnosis-specific cancer drugs. (Cancer Sci 2011; 102: 2206-2213

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.