An Azomethine Derivative, 1-(4-nitrophenyl)-N-phenylmethanimine (BCS2) Ameliorated 7,12-dimethylbenz(a)anthracene-induced Mammary Carcinoma through Nrf2-Keap1-HO-1 Pathway.

Aims: The aim of this study is the evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against mammary carcinoma through the Nrf2- Keap1-HO-1 pathway.

Background: The global prevalence of breast cancer is rising at an alarming rate. The facilitation of abnormal cell proliferation in mammary carcinoma occurs due to the disruption of signaling pathways that balance pro- and antioxidant status, thereby producing oxidative stress that disrupts genomic stability. Therefore, introducing a potent antioxidant molecule with antitumor activity is of paramount importance for treating breast cancer.

Objective: Synthesis, characterization, and in-vitro, in-vivo, and in-silico evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against chemical carcinogen- induced mammary carcinogenesis in Sprague-Dawley rats.

Methods: An azomethine derivative, 1-(4-nitrophenyl)-N-phenylmethanimine (BCS2), was synthesized and characterized based on its spectral data. The cytotoxic potential was observed on breast cancer cells, MCF-7, MDA-MB-231, and MDA-MB-468. The in vivo chemotherapeutic potential of BCS2 was established on 7,12-dimethylbenz(a)anthracene (DMBA) induced breast cancer in Sprague-Dawley (SD) rats. The effect of BCS2 on kelch-like ECH-associated protein- 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated-B (NF-κB) was evaluated through ELISA and qPCR techniques. Furthermore, the binding potential and stability of BCS2 with Keap-1, HO-1, and MAPK were predicted using in silico molecular docking and dynamics studies. Additionally, drug-likeness properties of BCS2 were evaluated using in silico ADMET tools.

Results: BCS2 showed remarkable cytotoxic activity on MCF-7 cells followed by MDA-MB- 231 and MDA-MB-468 cells having an IC50 of 2.368μM, 4.843μM and 6.472μM respectively, without affecting normal breast cells, MCF-10A. In the DMBA-induced animal model, BCS2 showed potent antitumor potential and showed protective action on endogenous-enzymatic and non-enzymatic antioxidants in cancer-bearing animals. Marked improvement in cellular architecture and ultrastructure of breast/tumor tissues excised from experimental animals was noted through histopathological and field emission scanning electron microscopy (FESEM) analyses. Significant upregulation of antioxidant proteins, Keap1 and HO-1, and downregulation of inflammatory proteins, MAPK, and NF-κB was observed after BCS2 treatment. The in silico computational studies predicted the potent binding of BCS2 with the active pockets of Keap1, HO-1, and MAPK proteins that validated the biological findings.

Conclusion: The study revealed BCS2's potent antioxidant and antitumor potential against mammary carcinoma through the Nrf2-Keap1-HO-1 signaling pathway.