br Active DUB labeling assays br Active
2.7. Active DUB labeling assays
Active DUB labeling assays was performed as previously described
(Liu et al., 2014). Briefly, 22RV1 cck-8 were treated with Aur for 24 h and the lysates were incubated with HA-UbVS for 1 h at 37 °C, which was followed by boiling in reducing sample buﬀer. Samples were finally analyzed using western blot.
RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to perform total RNA isolation from the cells as previously described (Huang et al., 2012). The concentration of purified total RNA and the 260:280 nm ratios were measured to evaluate the quality. Reverse transcription was performed to obtain cDNA from 1 µg total RNA using the kit from Ta-KaRa Biotechnology. Quantitative real time PCR was performed using SYBR Green PCR Master Mix reagent kits from Applied Biosystems Inc. The following PCR primers (Liao et al., 2017) were used: AR forward: 5′-GGT GAG CAG AGT GCC CTA TC-3′, AR reverse: 5′-GAA GAC CTT GCA GCT TCC AC-3′; PSA forward: 5′-AGG CCT TCC CTG TAC ACC AA-3′, PSA reverse: 5′-GTC TTG GCC TGG TCA TTT CC-3′; GAPDH forward: r> 5′-TCC CAT CAC CAT CTT CCA-3′, GAPDH reverse: 5′-CAT CAC GCC ACA GTT TCC-3’. Each sample was analyzed in triplicate.
2.9. Nude mouse xenograft model
The nude BALB/c mice (18–22 g, male) were purchased from Guangdong Animal Center as the experiment animals. All the animals were housed in an environment with temperature of 22 ± 1 °C, re-lative humidity of 50 ± 1% and a light/dark cycle of 12/12 h. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Guangzhou Medical University institutional animal care and conducted according to the AAALAC and the IACUC guidelines. Each mouse received a sub-cutaneous injection of 1 × 107 cells. After 72 h, the mice were ran-domly assigned into two groups receiving injections of either vehicle (10% DMSO, 30% Cremophor EL and 60% normal saline) or Aur (6 mg/ kg/day, i.p.), respectively for 2 weeks.
2.10. Statistical methods
The data from three independent experiments were expressed as mean ± standard deviation (SD). Graph Pad Prism software was used to perform statistical analyses, and data were analyzed using Student's t-test or ANOVA. The value of p < 0.05 was considered statistically significant.
3.1. Aur suppresses the growth of androgen receptor-positive PCa cells and PCa xenografts
Recent studies have revealed potent antitumor eﬀects of Aur in several types of cancer (Fiskus et al., 2014; Liu et al., 2014; Staﬀord et al., 2018). To evaluate the anti-tumor eﬀect of Aur on androgen receptor-positive PCa cells, in the present study we used two androgen receptor-positive cell lines: LNcap and 22RV1 cells. LNcap and 22RV1 cells were exposed to Aur (0–2 μmol/L) for various durations. MTS assay was used to evaluate cell viability. As shown in Fig. 1A and B, Aur remarkably suppressed the cell viability of androgen receptor-positive PCa cells (p < 0.05). Next, we detected the long-term proliferative ability of 22RV1 cells after treatment of Aur for 24 h, and found that the colony formation were significantly inhibited by Aur in a dose depen-dent fashion (p < 0.05) (Fig. 1E). The above data demonstrated the in vitro cell growth suppression eﬀect of Aur on androgen receptor-posi-tive PCa cells. The eﬀect of Aur on androgen receptor-negative PCa cells was also assessed using the above assay. We found that Aur induced the loss of cell viability also in PC3 and DU145 cells, but less eﬃciently than in LNcap or 22RV1 (Fig. 1C and D). European Journal of Pharmacology 846 (2019) 1–11
In order to further explore the in vivo eﬀect of Aur, xenograft models of 22RV1 cells were established in nude mice which were then treated with Aur every day (6 mg/kg/day, i.p). We found that the average tumor weight was 0.13 g in Aur-treated group while it was 0.22 g (p < 0.05) in the vehicle group; however, no significant dif-ference in body weight was evident between the Aur-treated group and the vehicle group (Fig. 1F-H). Subsequently, immunochemical staining revealed that the levels of androgen receptor and Ki67 were decreased but cleaved-caspase-3 was increased in xenograft tumor tissues treated by Aur (Fig. 1I). Together, these results demonstrate that Aur restrains the development of androgen receptor-positive PCa cells.
3.2. Aur induces apoptosis in androgen receptor-positive PCa cells
Next we tested whether Aur would trigger cell death of androgen receptor-positive PCa. Flow cytometry and fluorescence microscopy following Annexin V-FITC/PI staining were employed to quantify apoptotic cell death at both early and advanced stages. We observed that a significantly higher percentage of Annexin-V/PI-positive cells were detected in the Aur-treatment group, compared with the control treatment group; and Aur induced cell death in a dose-dependent manner (p < 0.05) (Fig. 2A-C). Recently, it has been reported that Aur induces apoptosis through caspase activation in various cancer cells (Fiskus et al., 2014; Liu et al., 2014). Next, we explored whether the induction of apoptosis by Aur in androgen receptor-responsive PCa cells would be related to caspase activation. As shown in Fig. 2D, Aur dra-matically induced caspase-3 activation and the cleavage of PARP in LNcap and 22RV1 cells.