9-PAN promotes tubulin- and ROS-mediated cell death in human triple-negative breast cancer cells

Objectives: To examine the antiproliferative effect of a rationally designed, novel noscapine analogue, 9-((perfluorophenyl)methylene) aminonoscapine, '9-PAN') on MDA-MB-231 breast cancer cell line, and to elucidate the underlying mechanism of action.

Methods: The rationally designed Schiff base-containing compound, 9-PAN, was characterized using IR, NMR and mass spectra analysis. The effect of the compound on cell viability was studied using an MTT assay. Cell cycle and cell death analyses were performed using flow cytometry. Binding interactions of 9-PAN with tubulin were studied using spectrofluorometry. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were investigated using the probes, DCFDA and rhodamine-123, respectively. Immunofluorescence imaging was used to visualize cellular microtubules.

Key findings: 9-PAN inhibited cell proliferation (IC₅₀ of 20 ± 0.3 µm) and colony formation (IC₅₀, 6.2 ± 0.3 µm) by arresting the cells at G₂/M phase of the cell cycle. It bound to tubulin in a concentration-dependent manner without considerably altering the tertiary conformation of the protein or the polymer mass of the microtubules in vitro. The noscapinoid substantially damaged cellular microtubule network and induced cell death, facilitated by elevated levels of ROS.

Conclusions: 9-PAN exerts its antiproliferative effect by targeting tubulin and elevating ROS level in the cells.

Keywords: MDA‐MB‐231; cell proliferation; microtubules; noscapine; reactive oxygen species.