Antiangiogenic and Antitumoral Activity of Phenyl-3-(2-Chloroethyl)Ureas A Class of Soft Alkylating Agents Disrupting Microtubules That Are Unaffected by Cell Adhesion-Mediated Drug Resistance

Centre de Recherche, Unité de Biotechnologie et de Bioingénierie, CHUQ, Hôpital Saint-François d'Assise, Québec, Canada.
Cancer Research (Impact Factor: 9.33). 08/2004; 64(13):4654-63. DOI: 10.1158/0008-5472.CAN-03-3715
Source: PubMed


The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

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Available from: Rene C.-Gaudreault
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    • "Phenyl-chloroethyl ureas (CEUs), resulting from the fusion of the chlorambucil aromatic moiety and the carmustine non-nitrosated pharmacophore, are developed as new antitumor drugs for chemotherapy. Several CEUs display cytotoxic activity in various cancer cell lines including those characterised by resistance to conventional chemotherapy (Gaudreault et al., 1994; Mounetou et al., 2001, 2003) and also in experimental tumour models (Lacroix et al., 1988; Miot-Noirault et al., 2004; Petitclerc et al., 2004). Interestingly , CEUs mediate their cytotoxicity by covalent binding to cell proteins, not by modifying the DNA, and the main alkylated protein for antimitotic CEUs is b-tubulin (Legault et al., 2000). "
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    ABSTRACT: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G(1)/S and G(2)/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. Proteins from B16 cells incubated with [(14)C-urea]-CCEU and [(125)I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but beta-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G(2) and G(1) phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G(1) phase. This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G(1)/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics.
    Full-text · Article · Nov 2007 · British Journal of Pharmacology
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    • "For biodistribution studies, the previously determined maximum-tolerated dose (MTD) of 13 mg kg À1 (Miot-Noirault et al, 2004) of [ 125 I]ICEU (activity per mouse: 74 KBq) were administered intraperitoneally (i.p.) to the animals when tumours had reached approximately 5 mm in diameter (day 10). For the antitumoral efficacy study, we used the previously published ICEU 'infraclinical protocol', since it was successful in inducing antitumour effects similar to those observed when using 5-fluorouracil (5-Fu) as positive control (Miot-Noirault et al, 2004; Petitclerc et al, 2004). N-(4-iodophenyl)-N 0 -(2-chloroethyl)urea was therefore injected i.p at the MTD of 13 mg kg À1 at days 1, 5 and 9 (n ¼ 13 animals), while mice in the control group (n ¼ 12) were sham treated. "
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    ABSTRACT: The antitumoral profile of the microtubule disrupter N-(4-iodophenyl)-N'-(2-chloroethyl)urea (ICEU) was characterised in vitro and in vivo using the CT-26 colon carcinoma cell line, on the basis of the drug uptake by the cells, the modifications of cell cycle, and beta-tubulin and lipid membrane profiles. N-(4-iodophenyl)-N'-(2-chloroethyl)urea exhibited a rapid and dose-dependent uptake by CT-26 cells suggesting its passive diffusion through the membranes. Intraperitoneally injected ICEU biodistributed into the grafted CT-26 tumour, resulting thus in a significant tumour growth inhibition (TGI). N-(4-iodophenyl)-N'-(2-chloroethyl)urea was also observed to accumulate within colon tissue. Tumour growth inhibition was associated with a slight increase in the number of G2 tetraploid tumour cells in vivo, whereas G2 blockage was more obvious in vitro. The phenotype of beta-tubulin alkylation that was clearly demonstrated in vitro was undetectable in vivo. Nuclear magnetic resonance analysis showed that cells blocked in G2 phase underwent apoptosis, as confirmed by an increase in the methylene group resonance of mobile lipids, parallel to sub-G1 accumulation of the cells. In vivo, a decrease of the signals of both the phospholipid precursors and the products of membrane degradation occurred concomitantly with TGI. This multi-analysis established, at least partly, the ICEU activity profile, in vitro and in vivo, providing additional data in favour of ICEU as a tubulin-interacting drug accumulating within the intestinal tract. This may provide a starting point for researches for future efficacious tubulin-interacting drugs for the treatment of colorectal cancers.
    Full-text · Article · Jul 2007 · British Journal of Cancer
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    • "Impairment of FA-dependent integrins clustering is not only an important aspect of anoikis signaling (Reddig and Juliano, 2005), through, for example, integrin-mediated death signaling, but also it is an underlying mechanism of cell adhesion mediateddrug resistance (CAM-DR) (Vachon et al., 2002; Harnois et al., 2004; Reddig and Juliano, 2005). In this context, it was shown that CEU, in contrast to cDDP, circumvent CAM-DR (Petitclerc et al., 2004). For example, we suggest that CEU and possibly other classical MT-disrupting agents interfere with CAM-DR-dependent survival signaling pathways by inducing the dismantlement of FA structures and thus blocking the integrins recruitment. "
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    ABSTRACT: Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH(2)-terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.
    Full-text · Article · Mar 2007 · Journal of Pharmacology and Experimental Therapeutics
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