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. 2023 Jan 20:14:1110146.
doi: 10.3389/fphar.2023.1110146. eCollection 2023.

Second generation androgen receptor antagonist, TQB3720 abrogates prostate cancer growth via AR/GPX4 axis activated ferroptosis

Affiliations

Second generation androgen receptor antagonist, TQB3720 abrogates prostate cancer growth via AR/GPX4 axis activated ferroptosis

Zhongqing Zhang et al. Front Pharmacol. .

Abstract

Purpose: Prostate cancer (PCa) poses a great threat to humans. The study aimed to evaluate the potential of TQB3720 in promoting ferroptosis to suppress prostate cancer, providing a theoretical basis for PCa therapy. Methods: PCa cells and nude mice models were divided into TQB3720, enzalutamide (ENZ), and control groups. Sulforhodamine B assay, colony formation assessment, organoids culture system, and the CCK8 assay were used for detecting proliferation. Western blot assay was processed to detect the expression of androgen receptor (AR), ferroptosis, and apoptosis-related genes. Flow cytometry was applied to measure the intracellular ROS levels. ELISA was performed to determine the cellular oxidized glutathione (GSSG) and malondialdehyde (MDA) levels. RT-qPCR was conducted to detect the mRNA expression of genes in AR signaling. BODIPYTM™ 581/591 was processed for detection of intracellular lipid peroxidation levels. The interaction of AR with other translational factor complex proteins was explored using Co-immunoprecipitation (Co-IP), and the chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of AR-involved translational complex to downstream genes promoter. Luciferase reporter assay was conducted to examine the translation activity of GPX4 promoter, and immunohistochemistry (IHC) was conducted to analyze the levels of c-MYC, Ki-67 and AR in TQB3720-treated cancer tissues. Results: Here, we found TQB3720 inhibits the growth of prostate cancer in vitro and in vivo. TQB3720 treatment induced intracellular levels of GSSG and MDA significantly, by which hints AR antagonist caused ferroptosis-related cell death. Moreover, molecular evidence shown TQB3720 regulates downstream of AR signaling by binding AR resulting in inhibition of AR entry into the nucleus. Additional, we also proved that TQB3720 abrogates the interaction between AR and SP1 and leads to decrease GPX4 transcription. Conclusion: TQB3720 promotes ferroptosis in prostate cancer cells by reducing the AR/SP1 transcriptional complex binding to GPX4 promoter. As a result, it is suggested to be a potential drug for clinic prostate cancer treatment.

Keywords: GPx4; TQB3720; androgen receptor; ferroptosis; prostate cancer.

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Conflict of interest statement

Author DY was employed by the company Chia Tai Tianqing Pharmaceutical Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
TQB3720 inhibited the growth of prostate cells (A–D) Sulforhodamine B assays were conducted to detect the effect of TQB3720 on the proliferation of LNCap95 and 22RV1 cells, # p < 0.01 vs. 24 h. (E) Effect of TQB3720 on colony formation of LNCap95, 22RV1 cells, # p < 0.01 vs. 24 h. (F) Organoid assay showing the effect of TQB3720 on organoid formation of clinic human prostate cancer primary cells.
FIGURE 2
FIGURE 2
TQB3720 induced ferroptosis of prostate cancer cells. (A,B) The proliferation of LNCap95 and 22RV1 cells significantly decreased following TQB3720 incubation for 48 h, whereas ferrostatin-1 and liprostatin-1 abolished this effect, # p < 0.01 vs. control, *p < 0.01 vs. TQB3720 (5 μM) (C,D) Western blot showed effects of TQB3720 on expression of ferroptosis marker GPX4, # p < 0.01 vs. control. (E,F) Flow cytometry assay showed the effects of TQB3720 on ROS levels using DCFH-DA probes, # p < 0.01 vs. control. (G,H) ELISA results showed the effects of TQB3720 on GSSG levels in cancer cells, # p < 0.01 vs. control. (I–J) ELISA assay proved that the effects of TQB7320 on MDA levels in cancer cells, # p < 0.01 vs. control.
FIGURE 3
FIGURE 3
TQB3720 induced ferroptosis via AR-GPX4 axis (A,B) RT-qPCR detected the effects of on expression of genes in AR signaling pathway, ferroptosis pathway, and apoptotic pathway, # p < 0.01 vs. control. (C,D) Western blot examined the effects of on expression of genes in AR signaling pathway, ferroptosis pathway, and apoptotic pathway, # p < 0.01 vs. control. (E,F) Western blot determined the expression of AR and GPX4 after knockdown of AR, # p < 0.01 vs. si-control. (G) The BODIPY 581/591 assay revealed the effects of AR knockdown on lipid peroxidation. Blue indicates the hochest stained nuclei, and red indicates lipid peroxidation levels. (H) The cells were transfected with HA-AR for 24 h after knocking down GPX4 for 48 h, and then they were incubated with 5 μM TQB3720 for another 48 h. The BODIPY 581/591 assay was used to determine the lipid peroxidation levels indicated by hochest-stained nuclei in blue and lipid peroxidation in red. (I) The cells were transfected with HA-AR for 24 h after knocking GPX4 down for 48 h and then incubated with 5 μM TQB3720 for another 48 h, and then, MTT assay was used to detect the cell proliferation, # p < 0.01 vs. control.
FIGURE 4
FIGURE 4
TQB3720 inhibited downstream of AR signaling by inhibiting AR entry into the nucleus (A, B) Western blot examined the effects of TQB3720 and ENZ on PSA expressions, and the protein band density was determined using ImageJ software, # p < 0.01 vs. control (absence of any indicated chemicals). (C,D) Western blot examined the effects of TQB3720 and ENZ on AR entry into nuclei from cytoplasm, and the protein band density was determined using ImageJ software, # p < 0.01 vs. control (absence of any indicated chemicals).
FIGURE 5
FIGURE 5
TQB3720 impaired GPX4 expression by inhibiting AR binding to SP1 (A, B) Co-IP assay showed the effect of TQB3720 on the interaction between AR and SP1 in LNcap95 and 22RV1. (C, D) RT-qPCR detected content of AR- or SP1-binding GPX4 promoter after performing ChIP assay, # p < 0.01 vs. control. (E) Luciferase reporter assay analyzed the impacts of TQB7320 on luciferase activity of GPX4 promoter, # p < 0.01 vs. control.
FIGURE 6
FIGURE 6
TQB3720 inhibited PCa carcinogenesis. (A, B) The tumor volume in each group of nude mice, # p < 0.01 vs. TQB3720 (0 mg/kg). (C) IHC examined the influences of TQB7320 on the expression levels of AR, Ki-67, # p < 0.01 vs. TQB3720 (0 mg/kg). (D) Western blot showed the impacts of TQB7320 on content of AR proteins in the nucleus or cytoplasm tumors from nude mice. (E) Western blot examined the effects of TQB7320 on expression of GPX4 protein in tumors.

References

    1. Bassetto M., Ferla S., Pertusati F., Kandil S., Westwell A. D., Brancale A., et al. (2016). Design and synthesis of novel bicalutamide and enzalutamide derivatives as antiproliferative agents for the treatment of prostate cancer. Eur. J. Med. Chem. 118, 230–243. 10.1016/j.ejmech.2016.04.052 - DOI - PubMed
    1. Chang M. T., Tsai L-C., Goto K. N., Lee K. H., Shyur L. F. (2022) Phyto-sesquiterpene lactones DET and DETD-35 induce ferroptosis in vemurafenib sensitive and resistant melanoma via GPX4 inhibition and metabolic reprogramming. Pharmacol. Res. 178, 106148. 10.1016/j.phrs.2022.106148 - DOI - PubMed
    1. Chen P. H., Tsao Y. P., Wang C. C., Chen S. L. (2008). Nuclear receptor interaction protein, a coactivator of androgen receptors (AR), is regulated by AR and Sp1 to feed forward and activate its own gene expression through AR protein stability. Nucleic Acids Res. 36 (1), 51–66. 10.1093/nar/gkm942 - DOI - PMC - PubMed
    1. Chen Y., Zhou Q., Hankey W., Fang X., Yuan F. (2022). Second generation androgen receptor antagonists and challenges in prostate cancer treatment. Cell Death Dis. 13 (7), 632. 10.1038/s41419-022-05084-1 - DOI - PMC - PubMed
    1. Cho D., Blasio C. J. D., Rhee A. C., Kattan M. W. (2003). Prognostic factors for survival in patients with hormone-refractory prostate cancer (HRPC) after initial androgen deprivation therapy (ADT). Urol. Oncol. 21, 282. 10.1016/s1078-1439(03)00057-7 - DOI - PubMed