br To further explore the
To further explore the mechanism by which Aur induces cancer cell death, we detected the expression of Bcl-2 protein, a major member of the Bcl-2 family. Bcl-2 is located on mitochondrial membrane and mostly regulates the permeability of mitochondrial membrane (Yamaguchi et al., 2018). The BCL-2 family exerts central roles in apoptotic pathways and regulates cancer cell's survival and death (Gross, 2016; Savitskaya and Onishchenko, 2015; Zan et al., 2016). We found that Aur triggered a downregulation of anti-apoptotic protein Bcl-2 in PCa cells, thus indicating that mitochondrial respiration is in-volved in cell death induced by Aur (Fig. 2D).
3.3. Aur triggers accumulation of ubiquitinated proteins and ER stress
Our previous study has suggested that Aur is a proteasomal DUB inhibitor targeting UCHL5 and USP14 (Liu et al., 2014). Here, we used a DUB suicide-substrate probe (HA-UbVS) to determine the DUB in-hibition capacity of Aur in PCa cells. We observed that UbVS-tagged USP14 and UCHL5 were notably decreased in Aur treated cck-8 com-pared with the vehicle control treated cells (Fig. 3A), indicating that the activity of USP14 and UCHL5 were inhibited by Aur.
To investigate the co-silencing eﬀect of USP14 and UCHL5 on prostate cancer cells, cell viability assay was performed to detect in 22RV1 and LNcap post either USP14 siRNA, UCHL5 siRNA or the combination treatment. It showed that co-silencing of USP14 and UCHL5 has synergistic eﬀect on cell proliferation(Fig. 3B). Next, we investigated the ability of Aur to inhibit the proteasome in androgen receptor-positive PCa cells by examining the expression of ubiquiti-nated proteins (Ub-prs) in LNcap and 22RV1 cells treated with Aur. We found that the K48-linked and total ubiquitinated proteins were in-creased by Aur treatment in a dose-dependent manner. Meanwhile, we found that phosphorylated eIF2α (P-eIF2α) and CHOP, both are related to endoplasmic reticulum (ER) stress, were significantly increased by Aur treatment in androgen receptor-positive PCa cells (Fig. 3C). These results suggest that USP14 and UCHL5 inhibition and ER stress are involved in Aur induction of apoptosis in androgen receptor-positive PCa cells.
Fig. 1. Auranofin (Aur) suppressed the growth of PCa cells. (A-D) Cell viability assay. LNcap, 22RV1, DU145 and PC3 cells were exposed to diﬀerent con-centrations of Aur for 24 h or 48 h. Cancer cell viability was analyzed using MTS assay. Data presented were from three independent experiments. Mean ± S.D. (n = 3). (E) Eﬀect of Aur on PCa cell colony formation. PCa cells were exposed to Aur for 24 h, and transplanted in 30% agarose for 10 days (F-H) Eﬀect of Aur on PCa xenografts. Tumor xenografts in nude mice were established with 22RV1 cells and treated with Aur for 14 days. Xenograft image, tumor size, tumor weight and body weight were shown. *P < 0.05 vs the control treatment group. (I) Immunohistochemistry staining assay was used to detect the protein expres-sion of AR, Ki67 and cleaved caspase-3 of indicated xenografts. Representative images were shown at a magnification of 200. Quantitative data were shown.*P < 0.05, #P < 0.01 vs the control treatment group.
Fig. 2. Aur induced apoptosis of PCa cells. (A-B) After the treatment of Aur, apoptotic cells were detected with Annexin V- FITC/PI staining followed by flow cytometry analysis. Representative images and quantification of cell death are shown. *P < 0.05, vs the control treatment group. (C) LNcap and 22RV1 cells were seeded into 24-well plates and treated with Aur. Annexin V - FITC/PI was used to incubate the cells for 30 min in dark. The stained cells were then recorded with an inverted fluorescence microscope. (D) PCa cells were exposed to Aur. Cell lysates were collected from the culture cells. PARP, cleaved caspase-3 and Bcl-2 were analyzed using western blot. Quantitative data were shown. GAPDH was used as a loading control. *P < 0.05, #P < 0.01 vs the control treatment group.
3.4. Aur inhibits cell cycle progression
The experiments described above investigated the antitumor eﬀect of Aur through growth suppression and induction of apoptosis signaling pathways. Given that the inhibition of USP14 arrests cell cycle through regulating key proteins related to the G0/G1 to S phase transition (Liao et al., 2017, 2018), we further explored the underlying mechanism by which Aur suppresses the growth of androgen receptor-positive PCa cells. Cell cycle progression in each group was monitored using flow cytometry in LNcap and 22RV1 cells. Notably, Aur treatment blocked cell cycle at the G0/G1 phase in both cell lines (Fig. 4A and B). Sub-sequently, we measured the level of proteins associated with regulation of cell cycle using western blot analyses. We found that expression le-vels of CDK4 and cyclin D1 were decreased while p21 protein expres-sion was increased by Aur treatment in PCa cells (Fig. 4C and D). It is well-known that CDK4 and cyclin D1 promote and p21 blocks G1-S phase transition and cell cycle progression. Hence, these results to-gether demonstrate that Aur induces G0/G1 cell cycle arrest by mod-ulating the expression of CDK4, cyclin D1 and p21.