Hepatocellular carcinoma (HCC) is the fifth most common form of cancer worldwide and the third most common Roscovitine ic50 cause of cancer-related deaths (Raza and Sood, 2014). Safe
and effective chemotherapeutic reagents such as DHA are needed for use against HCC, and it remains important to elucidate the cytotoxic mechanisms of DHA against HCC. As mentioned above, there have been several studies on the cytotoxic mechanisms of DHA and the p53-dependent inhibitory effects of PFT using experimental cell culture models, but it is unknown whether PFT affects DHA-induced cytotoxicity in human HCC cells. In this report, we examined the effects of PFT on DHA-induced reductions in cell survival in HepG2 cells, as well as the effects on p53 expression, oxidative stress, autophagy and mitochondrial damage. This is the first report to suggest that PFT acts via a p53-independent mechanism against DHA-induced cytotoxicity in HepG2 cells. Human hepatoma HepG2, Hep3B or Huh7 cells were supplied by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). Cells were routinely kept IDO inhibitor in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin G (100 U/ml)/streptomycin (100 μg/ml) at 37 °C in a humidified 5% CO2-95% air incubator under standard conditions. The drugs used in these
experiments, pifithrin-α (PFT) or cis-4, 7, 10, 13, 16, 19-DHA (#D2534, ≥98%; Sigma, St. Louis, MO) and all other reagents were of the highest grade available, and were supplied by either Sigma or Wako Pure Chemical Industries (Osaka, Japan). Cell culture reagents were obtained from Life Technologies™ (Carlsbad, CA). DHA was dissolved in ethanol and stored as a 200 mM stock solution, flushed with argon, in lightproof containers at −20 °C. Light exposure was kept to a minimum for all drugs used. All to antibodies using for Western blotting were purchased from Cell Signaling Technology (Danvers, MA). siRNA-p53 (si-p53) and siRNA-control (non-targeting siRNA; negative control [Neg]) were transfected into HepG2 cells using HyperFect transfection reagent (Qiagen, Valencia, CA) according to the protocol
supplied by the manufacturer. A non-targeting siRNA was used as a control for the non-sequence-specific effects of transfected siRNAs. The siRNAs (Qiagen) used were si-p53 from FlexiTube siRNA (catalog no. SI00011655) and negative control from AllStars Neg. Control siRNA (catalog no. SI03650318). Briefly, 5 × 104 HepG2 cells containing each siRNA (final concentration, 10 nM) and HyperFect reagent were incubated for 24 h for assessment of p53 expression or cytotoxic effects by DHA. In order to confirm knockdown by siRNA in HepG2 cells, expression levels of p53 messenger RNA (mRNA) (GenBank Accession no. NM_000546.5) were quantified by real-time polymerase chain reaction (qPCR) with Light Cycler (Roche, Basel, Switzerland).