br Auranofin Aur otherwise known as
Auranofin (Aur), otherwise known as a thioredoxin reductase in-hibitor (Fiskus et al., 2014; Rigobello et al., 2004), has also been re-ported as a USP14/UCHL5 selective inhibitor (Huang et al., 2016; Liu et al., 2014). Aur exerts potent anti-tumor eﬀects through regulating proteolytic activities of the 19 S. In our previous report, we have de-monstrated that Aur's thioredoxin reductase inhibition is not necessary for its proteasome inhibition and that proteasome inhibition is required for cytotoxicity mediated by Aur (Liu et al., 2014). Our prior studies also have shown that USP14 stabilizes androgen receptor by deubi-quitination in breast cancer and PCa (Liao et al., 2017, 2018), sug-gesting that Aur might be beneficial to PCa treatment. Since this pre-mise has not yet been examined, the aim of the present study was to fill this gap. By examining the eﬀect of Aur on PCa and exploring the un-derlying mechanisms, the present study provides further compelling support for a promising anti-cancer strategy in which inhibition of USP14 and UCHL5 with Aur suppresses the growth and progression of androgen receptor-positive PCa both in vitro and in vivo.
2. Material and methods
Auranofin (C20H35AuO9PS) was obtained from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and suspended in DMSO at 10 mmol/L concentration, aliquoted and stored at −80 °C. MG132 was purchased from Selleckchem (Houston, TX, USA). Antibodies were obtained from following sources: anti- androgen receptor (Abcam, USA); anti-Bcl-2 (15071s), anti-caspase cleave 3(9661 s), anti-PARP (9542s), anti-CDK4 (1279s), anti-cyclinD1 (29269), anti-p21 (2947P), anti-CHOP (2895 s), anti-eIF2α (53249D7D3), phospho-eIF2α (3398Sd9g8), anti-ubiquitin (3936s), anti-K48-linkage Specific Polyubiquitin (8081s) (Cell Signaling Technology, MA, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA). MTS assay (CellTiter 96 Aqueous One Solution reagent) was gained from Promega Corporation. Apoptosis Detection Kit was obtained from Keygen Company in Nanjing, China. Dynabeads antibody coupling kit was obtained from Life technologies.
2.3. Cell viability assay
According to previous description, MTS assay was used to test cell viability (Liao et al., 2017). When CHIR99021 were in the logarithmic stage of growth, PCa cells were plated in triplicate in 96-well plates. Cells were then treated with serial dilutions of auranofin (0–2 μmol/L) for 24 h or 48 h. At each time point, 20 μl of MTS reagent was added to each well and incubated for another 3 h at 37 °C. The absorbance at 490 nm was determined using a microplate reader.
2.4. Colony formation assay
This assay was performed as we previously described (Liao et al., 2018). LNcap and 22RV1 cells were exposed to auranofin for 24 h and then suspended in the 6-well plates. Aur-treated cells were planked containing 30% agarose and the medium was the same as described in cell culture assay. Cells were cultured in the same atmosphere for 14 days. Then, 1% crystal violet solution was used to stain living cells. The images were visualized using digital camera and the experiments were performed in triplicate.
2.5. Cell death and cell cycle assay
According to previous studies (Huang et al., 2010), apoptosis was detected through flow cytometry and inverted fluorescence microscopy. Cells were seeded into 6-cm dishes and treated as indicated. For flow cytometry, cells were dissociated with trypsin for 1 min and harvested, and then washed with 4 ℃ PBS thrice. Next, collected cells were re-suspended with 500 μl binding buﬀer, after which the 5 μl Annexin V-FITC and 5 μl PI were added and cells were incubated for 15 min. For the fluorescence microscopy, 5 μl Annexin V-FITC and 5 μl PI were added into the live cell culture dishes and incubated with the cells for 30 min in cell incubator before removal of the unbound dye. The stained cells were then imaged with an inverted fluorescence micro-scope.
For cell cycle assay, after the treatment of Aur, PCa cells were col-lected and washed with PBS thrice, resuspended with 500 μl PBS and 2 ml 70% ethanol and fixed at 4 °C overnight. Lastly, cells were washed twice and incubated with RNase A, PI and 0.2% Triton X-100 complexes for half an hour at 4 °C in dark.
2.6. Western blot and Co-immunoprecipitation (Co-IP)
This assay was performed as previously reported (Huang et al., 2017). RIPA lysis buﬀer (Thermo Scientific) supplemented with pro-tease inhibitors, phosphatase inhibitor and PMSF were used to extract total crude proteins from the cultured cells. For Co-IP, a kit for detec-tion of ubiquitinated proteins was used as previously reported (Liao et al., 2017). Firstly, antibodies of androgen receptor were coupled with dynabeads (Invitrogen) for 16–24 h. Lysates were extracted from LNcap cells exposed to auranofin and then added into antibodies-charged beads and mixed for 1 h. Then dynabeads were separated from the ly-sates and washed with lysis buﬀer. Lastly, the dynabeads suspended with SDS-containing blue loading buﬀer were incubated for 10 min at 70 °C. The proteins were subject to SDS-PAGE and transferred to PVDF membranes. Then membranes containing proteins were blocked with 5% defatted milk powder in phosphate buﬀer saline for one hour and washed with PBS-T for 5 min thrice. After incubating with primary antibodies overnight at 4 °C, secondary antibodies were used to in-cubate the membranes for 1 h at room temperature. Lastly, the bounded secondary antibodies were reacted with the ECL detection reagents and exposed to X-ray films.