(Z)-1,1-Dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane induces concentration-dependent growth inhibition, apoptosis, and coordinates regulation of apoptotic genes in TRAMP cells

Abstract

(Z)-1-1-Dichloro-2,3-diphenylcyclopropane (A(II)) and (Z)-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane [2-(4-methoxyphenyl)-A(II)] inhibit tubulin polymerization, PSA production, and the proliferation of human prostate cancer cells. The actions of the agents were studied in three transgenic adenocarcinomas of the mouse prostate (TRAMP) cell lines. Antiproliferative potencies were determined and cells treated with the more potent 2-(4-methoxyphenyl)-A(II) were examined for induction of apoptosis. Microarray analyses were conducted to determine the apoptosis-related genes up- and down-regulated by the agent. 2-(4-Methoxyphenyl)-A(II) concentration-dependently inhibited growth of all three cell lines. Fifty percent and 100% growth inhibitory and 50% lethal concentrations were determined to be 0.3, 1.5, and 5 muM, respectively. Minimum detectable apoptosis-inducing concentrations by ELISA were 0.10 to 0.14 muM. PARP cleavage and two-color flow cytometry assays verified apoptosis induction. Microarray analyses showed Bok and Siva-pending to be up-regulated and that Birc, Dad1, and Atf5 were down-regulated. 2-(4-methoxyphenyl)-A(II) inhibits proliferation and induces apoptosis in the in vivo-adaptable TRAMP cells, suggesting the compound should be further examined in preclinical models.

Fig. 1

Structures of test agents.

Fig. 2

Apoptosis due to 2-(4-methoxyphenyl)-A_II in the C1A, C2H, and C2N cell lines. (A) C1A cells. (B) C2H cells. (C) C2N cells. Cells were plated in 6-well plates at 20,000 cells/well. Cells were treated 24 hours after plating (time 0) with 2-(4-methoxyphenyl)-A_II at the GI₂₅, GI₅₀, GI₇₀, GI₇₆, and TGI concentrations of the agent. An ELISA assay for internucleosomal chromatin fragmentation was performed 24 hours after the test agent was added. The control cultures were treated with 0.2% DMSO. The minimum apoptosis-inducing concentrations of 2-(4-methoxyphenyl)-A_II determined by regression analysis of the concentration-response curves were 0.14 ± 0.05 µM for C1A cells, 0.10 ± 0.03 µM for C2H cells, and 0.12 ± 0.01 µM for C2N cells.

Fig. 3

Representative Western blot analysis of PARP cleavage in C1A cells treated with 1.5 µM 2-(4-methoxyphenyl)-A_II. Lysates from test agent- (T) and DMSO only-treated (C) cells at times of 0, 24, 48, and 72 hours were separated on 10% SDS-PAGE resolving gels, transferred to membrane and probed with PARP antibody. β-Actin was probed as a loading control. Three independent experiments were performed and variation was less than 10%.