Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway

Abstract

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.

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“…The fluorescence intensity was detected using a flow cytometer (BD Biosciences), and FITC annexin V+/PI- cell populations were considered apoptotic [ 55 ]. In order to quantify the phase distribution of the cell cycle, we stained cells with 40 μg/mL PI for 30 min and analyzed them by flow cytometry [ 56 ]. To assess the MMP (∆ Ψm ), we loaded cells with 10 μM JC-1 for 20 min, and the frequency of JC-1 aggregates and monomers were analyzed [ 57 ].…”

Inhibition of Lipopolysaccharide-Induced Inflammatory and Oxidative Responses by Trans-cinnamaldehyde in C2C12 Myoblasts

“…The cells were stained with 10 μM DCF-DA for 15 min in the dark at 37°C. The cells were then washed with PBS and immediately recorded at 480nm/520 nm by flow cytometry (BD Biosciences, San Jose, CA, USA) as previously described 41 . To compare the degree of ROS generation through fluorescence microscopic observation, the cells were stained with DCF-DA for 15 min at 37°C and then fixed with 3.7% paraformaldehyde for 10 min.…”

Sulforaphane Impact on Reactive Oxygen Species (ROS) in Bladder Carcinoma

“…SFN’s inhibitory activity is not restricted to bladder cancer but is apparent in other tumor types as well. Apoptosis induction by SFN via ROS is seen in hepatocellular carcinoma [ 155 ], lung cancer [ 156 ] and breast cancer cells [ 157 ]. How SFN specifically targets bladder cancer remains to be clarified.…”