Chemopreventive effect of arachidin-rich extract from peanut hairy root cultures on inflammation-induced cholangiocarcinogenesis in a cytokine-treated biliary cell model

Cholangiocarcinoma (CCA) is a highly prevalent malignancy in Southeast Asia and is strongly associated with chronic biliary inflammation. Preventive strategies targeting early inflammation-driven carcinogenic events are therefore of great interest. Arachidin-rich extracts derived from peanut hairy root cultures have demonstrated anti-inflammatory and antioxidant activities, suggesting their potential as chemopreventive agents. An in vitro model of inflammation-induced cholangiocarcinogenesis was established using human immortalized cholangiocytes (MMNK-1) treated with a pro-inflammatory cytokine mixture (IL-6, TNF-α, and IFN-γ). Cells were pretreated with a partially purified arachidin-rich extract prior to cytokine exposure. Activation of inflammatory signaling pathways (STAT3 and NF-κB), oxidative stress, inducible nitric oxide synthase (iNOS) expression, DNA damage, tumorigenic properties, and cell migration were evaluated. Molecular docking analysis was performed to explore the potential interaction between arachidin and STAT3. Cytokine treatment significantly induced STAT3 and NF-κB activation, increased intracellular reactive oxygen species, promoted DNA damage and enhanced tumorigenic and migratory properties of MMNK-1 cells. Pretreatment with arachidin-rich extract selectively suppressed STAT3 phosphorylation, reduced oxidative stress and iNOS expression, and attenuated DNA damage, colony formation, and cell migration. Molecular docking suggested a plausible interaction of arachidin within the STAT3 SH2 domain. These findings demonstrate that an arachidin-rich extract suppresses key inflammation-driven molecular and cellular events associated with early cholangiocarcinogenesis in a cytokine-treated biliary cell model. This study supports the potential of arachidin-rich extracts as chemopreventive agents against inflammation-mediated CCA, while highlighting the need for further in vivo and mechanistic validation.