Anti-proliferative and anti-inflammatory potential of 2-chloro-3-((4-hydroxyphenyl)amino)naphthalene-1,4-dione through targeting TNF-α/IL-8 and NF-κB signaling in HCT116 cancer cells: a comprehensive in silico docking and in vitro study

Colorectal cancer (CRC) is closely associated with chronic inflammation, and the NF-κB pathway acts as a central regulator of the inflammatory response that promotes tumor progression. In this study, we examined the anticancer and anti-inflammatory potential of anilino-1,4-naphthoquinone derivatives (compounds 3 , 8 , and 12 ) in CRC models using in vitro assays , in silico analysis with the Gold program, and molecular dynamics (MD) simulations. HCT116 cells were pre-exposed to these compounds before LPS stimulation. Cytotoxicity was determined using the MTT assay. The results indicated that compounds 3 , 8 , and 12 inhibited cell proliferation, leading to nuclear morphological changes, as evidenced by an increased sub-G1 population in HCT116 cells. In addition, Flow cytometry analysis showed that the compound significantly increased apoptotic cell death, particularly in the late-apoptotic population, compared with the control and reference treatments. Molecular docking analysis demonstrated that the tested compound favorably interacted with key amino acid residues within human NF-κB/p50, the p50/p65 NF-κB complex, and IKKβ, with binding patterns to the active sites comparable to those of the reference inhibitors, MLN120B and BMS-345541. In biological experiments, compound 12 reduced TNF-α and IL-8 secretion, indicating suppression of the NF-κB-mediated inflammatory pathway. Collectively, these findings suggest that compound 12 exerts anticancer activity through dual anti-inflammatory and pro-apoptotic mechanisms, possibly by modulating the NF-κB pathway. This compound may therefore represent a promising lead candidate for further drug development against CRC.