N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) as potential antineoplastic agents. Part 3: role of carbonyl groups in the covalent binding to the colchicine-binding site.

In the course of the development of N-phenyl-N'-(2-chloroethyl)ureas (CEUs) as potential antineoplastic agents, we investigated the effect of carbonylated substituting chains of the aromatic ring of CEU on their covalent binding to the colchicine-binding site (C-BS). In this study, we found that CEU, 5e, 5f, 8e, and 8f substituted by either a methyl ester or a methyl ketyl group at the omega-position exhibited a significant antiproliferative activity on HT-29, M21, and MCF-7 tumor cells. SDS-PAGE assays and cell cycle analysis confirmed that 5e, 5f, 8e, and 8f covalently bind to the C-BS and arrest the cell division in G(2)/M phase. Surprisingly, the presence of omega-carboxyl, omega-ethyl esters or omega-amides decreased significantly both the antiproliferative activity and the specificity toward beta-tubulin.

Women's decision making about the use of natural health products at menopause: a needs assessment and patient decision aid.

OBJECTIVE: To identify the decision-making needs of women about the use of natural health products (NHP) at menopause and to develop a decision aid responsive to their needs. DESIGN: A qualitative study using focus groups, key informant interviews and group consultation. Content analysis was guided by the Ottawa Decision Support Framework. METHODS: Six focus groups with menopausal women aged 45 to 64 (n = 40) and key informant interviews (n = 15; physicians, nurses, women' s advocacy group, NHP stores owners, pharmacists, policy makers) were conducted in two Canadian cities. Two groups of menopausal women (n = 11) were consulted to obtain feedback on the acceptability of the new patient decision aid. RESULTS: The most common difficult decisions identified by women were: whether or not to take NHP; which NHP to choose; and whether or not to take anything for menopausal symptoms. In addition, key informants identified the challenge of choosing between hormone therapy and NHP for menopausal symptoms. The main sources of difficulty in making these decisions were the following: (1) inadequate knowledge and unrealistic expectations associated with NHP; (2) closed mindedness of physicians to discussion about NHP; (3) conflicting opinions of others; (4) inadequate resources to support NHP decision-making (e.g., information, finances, time); and (5) menopausal symptoms interfering with decision-making (e.g., lack of sleep due to hot flashes). To facilitate decision making, participants suggested the need for information about available choices, tighter regulation of NHP by the government, and access to health professionals conversant in NHP and medical options. The patient decision aid was developed according to the International Patient Decision Aid Standards and based on women' s identified needs. Women described the aid as easy to understand and useful for considering the decisions about NHP. CONCLUSIONS: Middle-age women reported difficulty when facing decisions about the use of NHP. Many sources of difficulty could be addressed in the patient decision aid. Subsequent studies should evaluate the effect of this decision aid on the decision-making process of women.

New soft alkylating agents with enhanced cytotoxicity against cancer cells resistant to chemotherapeutics and hypoxia.

Chloroethylureas (CEU) are soft alkylating agents that covalently bind to beta-tubulin (betaTAC) and affect microtubule polymerization dynamics. Herein, we report the identification of a CEU subset and its corresponding oxazolines, which induce cell growth inhibition, apoptosis, and microtubule disruption without alkylating beta-tubulin (N-betaTAC). Both betaTAC and N-betaTAC trigger the collapse of mitochondrial potential (DeltaPsi(m)) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (rho(0)) and uncoupler of the mitochondrial respiratory chain (MRC) showed that betaTAC and N-betaTAC impaired the MRC. rho(0) cells displayed an increased sensitivity toward N-betaTAC as compared with rho(+) cells but, in contrast, were resistant to betaTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-betaTAC derivative, triggered massive swelling of isolated mitochondria. This effect was insensitive to cyclosporin A and to Bcl-2 addition. In contrast, adenine nucleotide translocator (ANT) antagonists, bongkrekic acid or atractyloside, diminished swelling induced by OXA-195. The antiproliferative activities of the N-betaTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by betaTAC and N-betaTAC. One of the proteins alkylated by N-betaTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that betaTAC and N-betaTAC, despite their common ability to affect the microtubule network, trigger different cytotoxic mechanisms in cancer cells. The role of mitochondria in these mechanisms and the potential of N-betaTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed.

Microtubule-destabilizing agents induce focal adhesion structure disorganization and anoikis in cancer cells.

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.

N-Phenyl-N'-(2-chloroethyl)ureas (CEU) as potential antineoplastic agents. Part 2: role of omega-hydroxyl group in the covalent binding to beta-tubulin.

Tubulin is the target of many anticancer drugs, including N-phenyl-N'-(2-chloroethyl)urea (CEU). Unlike most anti-beta-tubulin agents, CEUs are protein monoalkylating agents binding through their N'-(2-chloroethyl)urea moiety to an amino acid nearby the colchicine-binding site on beta-tubulin isoform-2. Following the previously synthesized and attractive N-(3-omega-hydroxyalkylphenyl)-N'-(2-chloroethyl)urea that exhibited growth inhibitory activity at the nanomolar level, we investigated the importance of lower alkyl and alkoxy groups to evaluate the effect of hydroxylated group and chain length on both cell growth inhibition and the mechanism of action of CEU. Here, we describe the preparation of two new series of CEU and show that the most potent CEU derivatives beside the omega-hydroxylated 1f were 2f and 3e, respectively. We have confirmed that the pentyl substituted CEUs 1f, 2f, and 3e are still covalently binding to beta-tubulin and still arrest cell division in G(2)/M phase.

Optimized N-phenyl-N'-(2-chloroethyl)ureas as potential antineoplastic agents: synthesis and growth inhibition activity.

In our ongoing research program aimed at the optimization of microtubule-self-assembly disrupting agents, we have prepared three series of phenylurea analogues (CEU), derived from N-(3-omega-hydroxyalkyl or 4-omega-hydroxyalkyl or 3-omega-hydroxyalkynyl)-phenyl-N'-(2-chloroethyl)ureas. Most compounds exhibit potent growth inhibitory activity on human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7 tumor cell lines, with a GI50 ranging from 250 nM to 8 microM. Among these new molecules, three CEUs exhibit GI50 in the nanomolar range. They are more potent by approximately an order of magnitude than previously described CEU analogues. As such, they are attractive hit compounds for the development of potent new alkylating antitubulin drugs.