One of the documented functions of NF-kB is its ability to promote cellular survival due to induction of specific genes that inhibit apoptotic machinery in both normal and malignant cells [11, 12]. NF-kB also prevents necrosis by inducing genes encoding antioxidant proteins [12–14]. Since

NF-kB is a usual pathway that promotes resistance to drugs and radiation by tumoural cells, inhibition of NF-kB seems to be LBH589 promising in improving the efficacy of conventional anti-cancer MK-2206 research buy therapies [15, 16]. NF-kB is also directly involved in oxidative stress and inflammation [12, 17]. N-acetylcysteine (NAC) is one of the most used antioxidant drugs in liver diseases [18, 19] and is known to be able to increase the levels of glutathione and also act as a free radical scavenger. Cell culture and animal studies have shown that BAY 11-7082 clinical trial NAC can

protect normal cells, but not malignant cells, from the toxic effects of radiotherapy and chemotherapy [20]. The administration of NAC may have a role in cancer prevention and even in the treatment of some forms of cancer, as DNA induced damage can be completely blocked by NAC [21, 22]. We herein tested the antitumoural effect of NAC on HCC cells and its relationship with the NF-kB pathway. Methods Cell culture and treatment Human HepG2 and Huh7 HCC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Stock cells were GPX6 routinely grown as monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum, penicillin (100 U/mL), streptomycin (100 mg/mL), glutamine (4 mM), and pyruvate (100 mg/mL) in a humidified 5% CO2 atmosphere at 37°C and the medium was changed every

other day. Cells were maintained in T75 culture flasks and subcultured once a week in a total volume of 10 mL of complete medium. Cell culture reagents were purchased from Gibco (Invitrogen, Carlsbad, CA, USA), and culture flasks and dishes were purchased from TPP (Techno Plastic Products, Switzerland). Twenty-four hours before treatments, 105 HepG2 and Huh7 cells were replated in 6-well plates containing IFN-α 2A (Blausiegel Ind Ltda, SP-Brazil) at concentrations ranging from 0 to 105 IU/mL and NAC (Sigma, Brazil) at final concentrations of 5, 10 and 20 mM. Both drugs were first diluted in PBS and then in DMEM to the final concentrations. Commercial p65 siRNA (250 mM) (Cell Signaling Biotechnology, Danvers, MA, USA) was used to suppress the NF-kB pathway, as described below. Cells were harvested after 24, 48, 72 and 96 h of treatment. Untreated cells used as controls (CO) were incubated in standard conditions. All experiments were performed in triplicate.