Ambrosin sesquiterpene lactone exerts selective and potent anticancer effects in drug-resistant human breast cancer cells (MDA-MB-231) through mitochondrial mediated apoptosis, ROS generation and targeting Akt/β-Catenin signaling pathway

Purpose: Breast cancer accounts for a significant proportion of cancer burden among women world over. Concerning breast cancer treatment, there are only few chemotherapeutic agents available, which also have serious side effects. The present study was thus designed to explore in vitro the antitumor effects of ambrosin sesquiterpene lactone against human drug resistant breast cancer cells (MDA-MB-231). Methods: WST-1 assay was used to determine cell viability. The fact that ambrosin induced apoptosis was studied through acridine orange (AO)/ethidium bromide (EB) staining using fluorescence microscopy as well as using flow cytometry in association with annexin-v/propidium iodide (PI) staining. Furthermore, western blot assay was used to study effects of ambrosin on apoptosis-related protein expressions including Bax and Bcl-2, as well as to study the effects on numerous caspases and Akt/beta-Catenin Signaling Pathway. The effects on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were evaluated by flow cytometry. Results: The results showed that ambrosin with an IC50 value of 25 mu M decreased the viability of the MDA-MB-231 cells. The cytotoxicity of ambrosin was also investigated on the MCF 12A normal breast cells which showed that it exerted very low toxic effects on these cells. Ambrosin also caused remarkable changes in the morphology and suppressed the colony forming potential of MDA-MB-231 cells. The AO/EB staining assay showed that ambrosin inhibits the viability of cancer cells via induction of apoptotic cell death which was associated with increase in Bax and reduction in Bcl-2 levels. The apoptotic cells increased from 3.5% in the controls to around 56% at 50 mu M concentration in the MDA-MB-231 cells. It was also seen that ambrosin treatment to these cancer cells resulted in substantial suppression in MMP and remarkable rise in ROS in a dose-dependent manner. This molecule also significantly inhibited the Akt/beta-catenin signalling pathway by reducing the expressions of phosphorylated GSK-3 beta and Akt. Conclusions: Taken all together, the results of our study indicate that ambrosin sesquiterpene may be developed as a promising anticancer agent in human breast cancer provided further in-depth studies are performed.