This study compared the sensitivity of human hepatoma Hep3B, SK-HEP-1, SMMC-7721, and BEL-7402 cells to cardiac steroids, including bufalin (BF), a bufalin derivative (BF211), ouabain (OUA), and digitoxin (DIG). Hep3B cells exhibited relatively low sensitivity to cardiac steroids. Expression levels of subunits of Na þ /K þ -ATPase were high in Hep3B cells. However, colocalization of Na þ /K þ -ATPase and caveolin was nearly undetectable in Hep3B cells. By using RNA-Seq technology, we found a

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... al of 36 genes to be differentially expressed between Hep3B cells and SK-HEP-1 cells, which are highly sensitive to cardiac steroids. Our bioinformatics analysis determined that these genes were mostly comprised of extracellular space, protein binding, and extracellular region. Among these 36 genes, apolipoprotein E (APOE) played a critical role, as knockdown APOE expression induced colocalization of Na þ /K þ -ATPase and caveolin and increased sensitivity of Hep3B cells to both proliferation-inhibiting and cytotoxic effects of BF or BF211. Also, the effects of BF on PI3K/AKT/GSK3b and apoptosis signal cascades were enhanced in APOE knockdown cells. The results of our study confirmed the role of Na þ /K þ -ATPase signalosome in cytotoxicity of cardiac steroids and suggested that APOE regulated the sensitivity of cells to cardiac steroids by affecting formation and function of Na þ /K þ -ATPase signalosome. In addition, intercellular interaction with high level of Na þ /K þ -ATPase b1 subunit may be also a factor in the low sensitivity of Hep3B cells to cardiac steroids.