Cellular targets of the anti-breast cancer agent Z-1,1-dichloro-2,3-diphenylcyclopropane: type II estrogen binding sites and tubulin.

Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, AII) is a known anti-breast cancer agent with apparent antiestrogenic effects and remarkably low toxicity in rodents. We have recently shown that AII and its major metabolite Z-alpha-chlorochalcone (ZCC) inhibit proliferation of both estrogen-responsive and -nonresponsive human breast cancer cells, suggesting its mechanism is not mediated by the type I estrogen receptor (ER). The present studies were performed to begin to define the molecular targets of AII and ZCC. Based on the compounds' structures and actions, we hypothesized that their effects could be due to interaction at type II estrogen binding sites (EBSII) and/or cellular microtubules. The affinities of AII ZCC and the positive control diethylstilbestrol (DES) for the ER (in MCF-7 and MCF-7/LY2 cells) and EBSII (in MCF-7, MCF 7/LY2, and MDA-MB231 cells) were determined with a whole cell assay for displacement of [3H]estradiol. The kinetics of their effects on cellular microtubules and cell cycle distribution of human breast cancer cells were measured by indirect immunofluorescence and flow cytometry. Their abilities to inhibit assembly of isolated tubulin in vitro were determined. AII, ZCC, and DES had similar affinities for the EBSII in the three cell lines. Neither AII nor ZCC displaced [3H]estradiol from the ER in MCF-7 cells, whereas DES did. The microtubule network of MDA-MB231 cells exposed to 100 microM AII or 10 microM ZCC began to disassemble within 1 hour of treatment and was completely diffuse after 6 hour of exposure to either drug. AII inhibited in vitro assembly of tubulin, with an IC50 of 6.7 +/- 0.9 microM, while ZCC was inactive below 40 microM. Both drugs caused accumulation of the cells in the G2/M phase of the cell cycle. The evidence suggests that the antitumor action of AII is mediated, at least in part, through the EBSII and/or perturbation of tubulin-microtubule dynamics. AII thus represents a new lead compound for design and discovery of novel antitumor agents directed against the EBSII and/or microtubules.