br Table br Individual Barriers and Facilitators
3.4. Aur inhibits Zinc protoporphyrin IX 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.
3.5. Aur downregulates the protein level of androgen receptor
Our previous studies have demonstrated that USP14 could regulate the stability of androgen receptor proteins in PCa and breast carci-nomas (Liao et al., 2017, 2018). Inhibition of USP14 significantly in-creased the degradation of androgen receptor protein. To verify whe-ther Aur can aﬀect the stability of androgen receptors via inhibiting UCHL5 and USP14, the androgen receptor protein levels were assessed using western blot. We found that Aur caused decreases in androgen receptor protein expression in androgen receptor-positive PCa cells in a dose-dependent manner (Fig. 5A and B). The androgen receptor sig-naling pathway mediates survival and growth of PCa via multiple me-chanisms. Recent studies have shown that a few DUBs can modulate the localization or function of target proteins. USP10 deubiquitinates an-drogen receptor in the cytoplasm and increases its nuclear import and transcriptional activity (Faus et al., 2005).
To better understand the expression of androgen receptors in the nucleus and in the cytoplasm and explore whether Aur could co-translocate with androgen receptors, we examined the androgen re-ceptor protein distribution and expression in LNcap and 22RV1 cells exposed to Aur (1, 2 μM) using confocal microscopy. As shown in
Fig. 3. Aur induced proteasome inhibition and ER stress in PCa cells. (A) Cell lysates were extracted from 22RV1 cells treated with or without Aur for 24 h and the lysates were incubated with the HA-UbVS probe for 1 h. The protein levels of native USP14 and UCHL5 as well as their UbVS-bound counterparts were im-munodetected using the primary antibodies against USP14 and UCHL5, respectively. Quantitative data were shown. GAPDH was used as an internal control. *P < 0.05, #P < 0.01 vs the control treatment group. (B) Cell viability analysis was performed on prostate cancer cells post either USP14 siRNA, UCHL5 siRNA or the combination treatment for 48 h.(C) 22RV1 and LNcap cells were exposed to the indicated concentrations of Aur for 24 h. Total proteins were extracted from the treated cells. Western blot assay was used to assess the total ubiquitinated proteins (Ub-prs.), K48-linked ubiquitin chain conjugated proteins (K48-), eIF2α, phosphorylated eIF2α (P-eIF2α) and CHOP expression. Quantitative data were shown. GAPDH was used as a loading control. *P < 0.05, #P < 0.01 vs the control treatment group.
Fig. 5C, androgen receptor was located in both the nucleus and cyto-plasm, albeit mainly in nucleus; Aur decreased the abundance of an-drogen receptor in the nucleus and cytoplasm but did not seem to alter androgen receptor nucleus to cytoplasm ratio. These results demon-strate that Aur downregulates the abundance of the total androgen receptor in androgen receptor-positive PCa cells.