A novel synthetic iminoquinone, BA-TPQ, as an anti-breast cancer agent: in vitro and in vivo activity and mechanisms of action

Abstract

Herein we report our examination of the anti-breast cancer activity of a novel synthetic compound, 7-(benzylamino)-1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one (BA-TPQ). This agent is an analog of a naturally-occurring marine compound, and was found to be the most active out of more than 40 related compounds. We investigated the in vitro activity of BA-TPQ on the survival, proliferation, and apoptosis of breast cancer cells using the MTT and BrdUrd assays, and Annexin/Annexin-PI staining and flow cytometry. The in vivo anti-cancer effects of BA-TPQ were evaluated in xenograft models of breast cancer. Finally, the mechanisms of action of the compound were also assessed by cDNA microarrays, RT-PCR and Western blotting. In a dose-dependent manner, BA-TPQ inhibited cell growth and induced apoptosis and cell cycle arrest in human MCF-7 and MDA-MB-468 breast cancer cells in vitro, and showed in vivo efficacy in mice bearing MCF-7 or MDA-MB-468 xenograft tumors. We demonstrated that BA-TPQ modifies the expression of numerous molecules involved in cell cycle progression and apoptosis. Similar changes in protein expression were observed in vitro and in vivo, as determined by examination of cells and excised xenograft tumors. Our preclinical data indicate that BA-TPQ is a potential therapeutic agent for breast cancer that has multiple hormone-, Her2- and p53-independent mechanisms of action, providing a basis for further development of the compound as a novel anticancer agent.

The pyrido[b]indole MDM2 inhibitor SP-141 exerts potent therapeutic effects in breast cancer models

“…The effects of the test compound on human breast cancer cell growth and viability, expressed as the percentage of cell survival, were determined using the MTT assay as described previously [54][55][56] . Cells were grown in 96-well plates at 3-4 Â 10 3 cells per well, and were exposed to the test compound (0-25 mM) for 72 h. After incubation with the compound, 10 ml of the MTT solution (5 mg ml À 1 ; Sigma-Aldrich Co.) were added to each well.…”

“…The effects of SP-141 on cell proliferation were determined using the BrdUrd incorporation assay (Oncogene, La Jolla, CA, USA) as reported previously [54][55][56] . In brief, cells were seeded in 96-well plates (3-5 Â 10 3 cells per well) and incubated with various concentrations of SP-141 (0, 0.1, 0.2, 0.5 and 1 mM) for 24 h. BrdUrd was added to the medium 10 h before termination of the experiment.…”

“…Cells in early and late stages of apoptosis were detected using an Annexin V-FITC apoptosis detection kit from BioVision (Mountain View, CA, USA) according to the manufacturer's protocol, as described previously [54][55][56] . In this procedure, 2-3 Â 10 5 cells were exposed to the test compound (0, 0.2, 0.5 and 1 mM) and incubated for 48 h before the analysis.…”

“…To determine the effects of the compound on the cell cycle distribution using the method described previously [54][55][56] , cells (2-3 Â 10 5 per well) were exposed to the test compound (0, 0.2 or 0.5 mM) and incubated for 24 h before the analysis. The cells were then trypsinized, washed with PBS and fixed in 1.5 ml of 95% ethanol at 4°C overnight, followed by incubation with RNAse and staining with propidium iodide (Sigma-Aldrich Co.).…”

“…For the in vitro studies, cells were exposed to various concentrations of SP-141 for 24 h. In vivo tissue homogenates were prepared in RIPA buffer (100 mg tumour tissue per 1 ml RIPA) for the immunoblotting analysis. Cell lysates were fractionated with identical amounts of protein using SDS-PAGE and transferred to Bio-Rad trans-Blot nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) as described previously [54][55][56] . The nitrocellulose membrane was then incubated in blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk) for 1 h at room temperature.…”