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 (beta TAC) 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-beta TAC). Both beta TAC and N-beta TAC trigger the collapse of mitochondrial potential (Delta Psi(m)) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratorydeficient cells (p) and uncoupler of the mitochondrial respiratory chain (MRC) showed that beta TAC and N-beta TAC impaired the MRC. p(0) cells displayed an increased sensitivity toward N-beta TAC as compared with p(+) cells but, in contrast, were resistant to beta TAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-OTAC 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-beta TACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by beta TAC and N-OTAC. One of the proteins alkylated by N-OTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that beta TAC and N-beta TAC, 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-beta TAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed.