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Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

ABSTRACT: Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and synthesized isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and rationally combined ITC-ARi with GSH synthesis inhibitor buthionine sulfoximine (BSO) to efficiently downregulate AR/AR splice variant and induce ferroptosis in CRPC cells. The representative ITC-ARi 13 is an AR ligand that contains an N-acetyl cysteine-masked ITC moiety and gradually releases parental unconjugated ITC 12b in aqueous solution. The in vitro anti-PCa activities of 13, such as growth inhibition and AR downregulation, are significantly enhanced when combined with BSO. The drug combination caused notable lipid peroxidation and the cell viability was effectively rescued by iron chelator, antioxidants or the inhibitor of heme oxygenase-1, supporting the induction of ferroptosis. 13 and BSO cooperatively downregulate AR and induce ferroptosis likely through increasing the accessibility of 13/12b to cellular targets, escalating free intracellular ferrous iron and attenuating GSH-centered cellular defense and adaptation. Further studies on the combination of ITC-ARi and GSH synthesis inhibitor could result in a new modality against CRPC.

SUBMITTER: Qin Z

PROVIDER: S-EPMC8168342 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

Qin Zhihui Z Ou Siyu S Xu Liping L Sorensen Kathleen K Zhang Yingxue Y Hu Dan-Ping DP Yang Zhe Z Hu Wen-Yang WY Chen Fei F Prins Gail S GS

Chemical biology & drug design 20210126 5

Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and synthesized isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and rationally combined ITC-ARi with GSH synthesis inhibitor buthionine sulfoximine (BSO) to efficiently downregulate AR/AR splice variant and induce ferroptosis in CRPC cells. The representative ITC-ARi 13 is an AR ligand that contains an N-acetyl cysteine-m ...[more]

PMID: 33470049

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Project description: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.

Project description:BackgroundEndothelial dysfunction caused by low androgen levels in penile tissue can lead to erectile dysfunction. The exact mechanism of endothelial dysfunction has not been thoroughly studied.ObjectiveThe study sought to verify whether low androgen levels induce ferroptosis of endothelial cells in rat penile tissue.MethodsRat penile cavernous endothelial cells (CP-R133) were divided into a no-androgen group (Dihydrotestosterone (DHT): 0 nmol/L), very low-androgen group (DHT: 0.1 nmol/L), low-androgen group (DHT: 1 nmol/L), DHT = 10 nmol/L group, DHT (0 nmol/L) + ferrostatin-1 (Fer-1) group, DHT (0.1 nmol/L) + Fer-1 group, DHT (1 nmol/L) + Fer-1 group, DHT (10 nmol/L) + Fer-1 group. Cell viability, intracellular ferrous ion (Fe2+), malondialdehyde (MDA), GSH into oxidized glutathione (GSSG), reactive oxygen species (ROS), nitric oxide (NO), transferrin receptor 1 protein (TfR1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), endothelial nitric oxide synthase (eNOS), and phospho-eNOS (p-eNOS) were detected.OutcomesLow androgen levels could induce ferroptosis of rat penile cavernous endothelial cells in vivo by upregulating the expressions of TfR1 and ACSL4 and downregulating the expressions of SLC7A11 and GPX4.ResultsCell viability, the levels of glutathione (GSH), NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 0 nmol/L group were lower than those in the other groups (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 0 nmol/L group were higher than those in the other groups (P < .05). Cell viability and the levels of GSH, NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 1 nmol/L group were lower than those in the DHT (1 nmol/L) + Fer-1 group, DHT = 10 nmol/L group, and DHT (10 nmol/L) + Fer-1 group (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 1 nmol/L group were higher than those in the DHT (1 nmol/L) + Fer-1 group, DHT = 10 nmol/L group, and DHT (10 nmol/L) + Fer-1 group (P < .05).Clinical implicationsA ferroptosis inhibitor might be a novel drug for treating erectile dysfunction caused by low androgen level.Strengths and limitationsThe results of this study need to be further confirmed in in vitro and in human studies. Meanwhile, further investigation is needed to clarify whether low androgen levels affect ferroptosis of rat penile cavernous smooth muscle and nerve cells.ConclusionLow androgen levels can induce ferroptosis of endothelial cells in rat penile tissue. Inhibition of ferroptosis can reverse endothelial dysfunction caused by low androgen levels.

Project description:Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15-35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20-1.89 in models of TNBC with high AR expression. AR-negative (AR-) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 μM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an in vivo AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

Project description:BackgroundCastration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC).MethodsWe employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation.ResultsAs expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines.ConclusionJJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

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Dataset Information

Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

Publications

Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

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