br Interestingly inhibition of both
Interestingly, inhibition of both USP14 and UCHL5 induced by auranofin and b-AP15 suppresses the proteasome function in multiple cell types and in vitro experiments, while sole inhibition of USP14 in-duced by IU1 actually enhances the proteasome activity, indicating that proteasome inhibition eﬀect only caused by inhibition of both USP14 and UCHL5, not only USP14, and that USP14 appears to have a non-catalytic association with proteasome as well. Most of ubiquitinated proteins were accumulated after proteasome inhibition caused by USP14 and UCHL5 inhibition. However, for a specific protein, such as AR, the inhibition of USP14 would impair the deubiquitination of AR, which consequently leads to the degradation of this protein. Additionally, this study found an unknown interesting eﬀect that aur-anofin also downregulates the mRNA level of AR. We were prompted to study the transcriptional inhibition of AR by auranofin in the future work.
In summary, the present study demonstrates that auranofin sup-presses the growth of LNcap and 22RV1 cells and induces high levels of cancer cell death. This most likely occurs through USP14 and UCHL5 inhibition which in turn suppresses androgen receptor transcription, destabilizes androgen receptor proteins, induces ER stress, abrogates cck8 progression, and causes ultimately apoptosis in PCa cells. These findings provide a promising new strategy for chemotherapy of androgen receptor-positive PCa.
Fig. 6. Aur inhibited the deubiquitination and transcription of androgen receptor in PCa cells. (A-B) Cycloheximide chase assay. PCa cells were exposed to cy-cloheximide (50 μg/ml) alone or to both cycloheximide and Aur (1 μM) for 0, 3, 6, 12 h. The collected protein lysates were assessed for androgen receptor expression using western blot assays. Representative images and densitometry data are shown. (C) LNcap cells were treated with Aur (1 μM) for 24 h and MG132 (10 μM) for 6 h before harvested. Total proteins were extracted from the cells. Co-IP assay was performed using androgen receptor antibody beads. When immunoprecipitated with androgen receptor antibody beads was performed or not, immunoblotted for ubiquitin and androgen receptor were analyzed. GAPDH was considered as a loading control. (D) The total RNAs were collected from the PCa cells after treatment of Aur for 12 h and were subject to RT-qPCR analysis for androgen receptor and PSA mRNA expression. Mean ± S.D. (n = 3). *P < 0.05, #P < 0.01 vs the control treatment group. (E) A proposed model for the inhibition of cell growth by Aur in androgen receptor-positive PCa cells.
CRediT authorship contribution statement
Ningning Liu: Conceptualization, Data curation, Methodology. Zhiqiang Guo: Conceptualization, Data curation, Methodology. Xiaohong Xia: Conceptualization, Data curation, Methodology. Yuning Liao: Conceptualization, Data curation, Methodology. Fangcheng Zhang: Conceptualization, Data curation, Methodology. Chuyi Huang: Conceptualization, Data curation, Methodology. Yuan Liu: Conceptualization, Data curation, Methodology. Xiumei Deng: Conceptualization, Data curation, Methodology. Lili Jiang: Conceptualization, Data curation, Methodology. Xuejun Wang: Writing- review & editing. Jinbao Liu: Conceptualization, Data curation, Funding acquisition, Methodology, Supervision, Writing- original draft, Writing- review & editing. Hongbiao Huang: Conceptualization, Data curation, Funding acquisition, Methodology, Supervision, Writing- original draft, Writing- review & editing. r> Acknowledgements
The study was supported by the National Natural Science Foundation of China (81472390, 81773213), The National Funds for Developing Local Colleges and Universities (B16056001), Natural Science Foundation Research Team of Guangdong Provincial (2018B030312001), the Science and Technology Program of Guangzhou (201604020001), Innovative Academic Team of Guangzhou Education System (1201610014), General Project (1201610098) from Guangzhou Education Commission, the Natural Science Foundation of Guangdong Province (2017A030313796), Guangzhou Health and Family Planning Science and Technology pro-ject (20181A011066), a Polarity Research Award Fund for Outstanding Young Teachers in Guangdong Provincial Higher Education Institutions (YQ2015136), the Project of Department of Education of Guangdong Province (2016KTSCX118, 2016KTSCX119), the Research Team of Department of Education of Guangdong Province (2017KCXTD027).
Conflict of interest
The authors declare no conflict of interest.
Bosco, C., Bosnyak, Z., Malmberg, A., Adolfsson, J., Keating, N.L., Van Hemelrijck, M., 2015. Quantifying observational evidence for risk of fatal and nonfatal cardiovascular disease following androgen deprivation therapy for prostate cancer: a meta-analysis. Eur. Urol. 68, 386–396.