Induction of human breast cancer cell apoptosis from G 2/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane

Abstract

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, A II) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of A II. MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 μM A II when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1–2 Mb fragments appeared after 6 hr, while 30–50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30–50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that A II blocks MDA-MB-231 cells in G 2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A II demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, “normal” human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that A II caused stimulation into S phase, and that G 2/M was the phase of the cell cycle from which cells apoptosed. A II caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited A II-induced cell death, indicating that its toxicity requires de novo protein synthesis.