Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison

Abstract

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time-dependent cell death in the MCF-7 and MDA-MB231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (<10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but <20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.

Introduction

A number of compounds that have either proven or potential activity against various diseases, including cancer, have been derived from marine organisms. The substituted pyrroles, which were synthesized based on similarity to marine derived compounds [1], [2] have previously been demonstrated to have significant growth inhibitory activity against a variety of human tumor cell lines, with antiproliferative effects evident at nanomolar concentrations in some human breast tumor cell lines [3], [4], [5], [6].

The development of synthetic or semisynthetic derivatives of the pyrroles is facilitated due to the relative ease with which these compounds can be synthesized and their functional groups manipulated [3]. One of these pyrroles, termed JG-03-14 (2,4-dibromo-5-carbethoxy-3-(3,4-dimethoxyphenyl) pyrrole), was found to act as a microtubule poison and to bind at the colchicine-binding site of tubulin [7] (and unpublished data).

The current investigation was designed to characterize the action of the substituted pyrrole, JG-03-14, in two human breast cancer cells lines expressing either wild type p53 (MCF-7 cells) or mutant p53 (MDA-MB231 cells). Our studies focused on the capacity of JG-03-14 to promote irreversible growth arrest and/or cell death, as well as to characterize the nature of the growth arrest/cell death response. We were also interested in evaluating the possibility that this compound would be effective in a tumor cell line expressing the multidrug resistant phenotype.