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

Abstract

Chloroethylureas (CEU) are soft alkylating agents that covalently bind to β-tubulin (βTAC) 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 β-tubulin (N-βTAC). Both βTAC and N-βTAC trigger the collapse of mitochondrial potential (ΔΨ m) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (ρ0) and uncoupler of the mitochondrial respiratory chain (MRC) showed that βTAC and N-βTAC impaired the MRC. ρ0 cells displayed an increased sensitivity toward N-βTAC as compared with ρ+ cells but, in contrast, were resistant to βTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-βTAC 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-βTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by βTAC and N-βTAC. One of the proteins alkylated by N-βTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that βTAC and N-βTAC, 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-βTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed. ©2007 American Association for Cancer Research.