Effects of JS-K, a novel anti-cancer nitric oxide prodrug, on gene expression in human hepatoma Hep3B cells

JS-K is a novel Anticancer nitric oxide (NO) prodrug effective against a variety of Cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate Anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100μM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100μM, while the LC₅₀ for JS-K was about 10μM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10μM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated Apoptosis pathways in Hep3B cells, including induction of Caspase-3, caspase-9, Bax, TNF-α, and IL-1β, and immunostaining for Caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-Myc in Hep3B cells. Thus, multiple molecular events appear to be associated with Anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to Apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration.