An Azomethine Derivative, BCS3, Targets XIAP and cIAP1/2 to Arrest Breast Cancer Progression Through MDM2-p53 and Bcl-2-Caspase Signaling Modulation

<b>Background</b>: Breast cancer influences more than 2 million women worldwide annually. Since apoptotic dysregulation is a cancer hallmark, targeting apoptotic regulators encompasses strategic drug development for cancer therapy. One such class of apoptotic regulators is inhibitors of apoptosis proteins (IAP) which are a class of E3 ubiquitin ligases that actively function to support cancer growth and survival. <b>Methods</b>: The current study reports design, synthesis, docking analysis (based on binding to IAP-BIR3 domains), anti-proliferative and anti-tumor potential of the azomethine derivative, 1-(4-chlorophenyl)-N-(4-ethoxyphenyl)methanimine (<b>BCS3</b>) on breast cancer (in vitro and in vivo) and its possible mechanisms of action. <b>Results</b>: Strong selective cytotoxic activity was observed in MDA-MB-231, MCF-7, and MDA-MB-468 breast cancer cell lines that exhibited IC50 values, 1.554 µM, 5.979 µM, and 6.462 µM, respectively, without affecting normal breast cells, MCF-10A. For the evaluation of the cytotoxic potential of <b>BCS3</b>, immunofluorescence, immunoblotting, and FACS (apoptosis and cell cycle) analyses were conducted. <b>BCS3</b> antagonized IAPs, thereby causing MDM2-p53 and Bcl-2-Caspase-mediated intrinsic and extrinsic apoptosis. It also modulated p53 expression causing p21-CDK1/cyclin B1-mediated cell cycle arrest at S and G2/M phases. The in vitro findings were consistent with in vivo findings as observed by reduced tumor volume and apoptosis initiation (TUNEL assay) by IAP downregulation. <b>BCS3</b> also produced potent synergistic effects with doxorubicin on tumor inhibition. <b>Conclusions</b>: Having witnessed the profound anti-proliferative potential of <b>BCS3</b>, the possible adverse effects related to anti-cancer therapy were examined following OECD 407 guidelines which confirmed its systemic safety profile and well tolerability. The results indicate the promising effect of <b>BCS3</b> as an IAP antagonist for breast cancer therapy with fewer adverse effects.