The
thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx
reductase (TrxR) was overexpressed in various types of human
cancer cells indicating that the Trx-TrxR system may be a potential target for anti-
cancer drug development. This study investigated the synergistic effect of
auranofin, a TrxR-specific inhibitor, on
sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that
sulforaphane significantly enhanced
auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of
sulforaphane and
auranofin on apoptosis was evidenced by an increased
annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of
caspases (-3, -8, and -9) and the degradation of
poly (ADP-ribose) polymerase, a substrate
protein of activated
caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of
reactive oxygen species (ROS). However, treatment with
N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by
auranofin and
sulforaphane. Furthermore, apoptosis induced by
auranofin and
sulforaphane was significantly increased through inhibition of the
phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of
auranofin and
sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.