Humic acid (HA), a group of high-molecular weight polymer, resulting from the decomposition of organic matter has been implicated as a possible etiological factor for Blackfoot disease and cancer. In this study, we evaluate the promotion effect of HA on the transformation in mouse epidermal JB6 clone 41 (JB6 Cl41) cells that have been used to identify the tumor promoting activity of various compounds. Our preliminary assay demonstrated that JB6 Cl41 cells with the treatment of HA at the concentration of 100 μg/ml for 72 and 96 h significantly increased reactive oxygen species (ROS) as compared to the untreated control. In addition, the 48 h cultured cells with HA pretreatment for 48 h also increased ROS as compared to the untreated control. HA-pretreated cells develop highly scattered and spindle-shaped cells with few observable cell-cell contacts, and contain more filopodia. In vitro wound-healing assay showed that JB6 Cl41 cells with HA pretreatment increased the migrating growth. Furthermore, transformed foci of JB6 Cl41 cells following the HA pretreatment were observed after 6 weeks culture. In anchorage-independent growth assay, we found that HA promoted the colony formation and that colonies were inhibited by antioxidant N-acetyl cysteine (NAC). Our results suggest that HA may promote the transformation of epidermal cells and that this process is mediated by the generation of ROS.
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