Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: Screening and identification of a novel small molecule inhibitor targeting AR. (A) Pyramid chart of the screening inhibitors of ARfl and ARv7 from the small molecule compounds library. (B) Three parental compounds were obtained by computer-aided drug design. (C) Effects of the top 8 compounds on cell viability in 22RV1 cells ( n = 3). (D–F) LNCaP (ARfl positive cells), 22RV1 (both ARfl and ARv7 positive cells) and PC3 (transfected with ARfl and ARv7 expressing plasmids) cells were administrated with the top eight compounds (10 μmol/L, 48h). Moreover, the protein levels of ARfl, ARv7 and PSA were analyzed by Western blot. (G) The chemical structure of LLU-206. (H) Binding model of LLU-206 in AR-LBD binding pocket. (I) The docking prediction of LLU-206 (purple) with AR-LBD (blue). (J) The interaction between AR-LBD protein and LLU-206 was measured by biacore. Results are expressed as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s., not significant.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: Transfection, Expressing, Western Blot, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: LLU-206 inhibits the proliferation and promotes the apoptosis of PCa and CRPC cells. (A) IC 50 values of LLU-206 and ENZ on cell viability in different PCa cells treated for 72 h (LNCaP, 22RV1, LNCaP-ENZR and LNCaP-ARfl OE ). (B) The viability of LNCaP cells was evaluated after exposure to LLU-206 treatment at different concentrations with or without stimulation of DHT (10 nmol/L, 72h), while ENZ, ARN-509 and ODM-201 were used as control. (C) 22RV1 cells were treated with the indicated concentrations of LLU-206 and 10 μmol/L ENZ for seven days, and the colony formation assay was counted and quantified. (D) After being cultured for 9 days, the spheres were treated with 10 μmol/L LLU-206, ODM-201, ARN-509 and ENZ for another five days. The spheroids were recorded on Days 11, 13 and 14. (E) The CD44 mRNA expression of the spheroids was detected by qPCR. (F) The apoptosis of 22RV1 cells was determined by flow cytometry after treatment with LLU-206 and ENZ (10 μmol/L, 48 h). (G) GSEA analysis of the RNA sequence for KEGG_CELL_CYCLE in 22RV1 cells treated with LLU-206 (10 μmol/L, 24 h) and control (left). After 22RV1 cells were treated with LLU206 or ENZ (10 μmol/L, 48 h), the cell cycles were detected by flow cytometry (right). (H) The apoptosis markers cleaved caspase3, Bcl-2, Bax; proliferation makers AKT and p-AKT, ARfl and ARv7 were analyzed by Western blot in 22RV1 cells treated with LLU-206 or ENZ for 48 h. Scale bar = 100 μm. Results are expressed as mean ± SEM, n = 3. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s., not significant.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: Control, Colony Assay, Cell Culture, Expressing, Flow Cytometry, Sequencing, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: LLU-206 suppresses the growth of 22RV1 and LNCaP-ENZR xenografts in vivo . (A) LNCaP ENZR cells were generated by culturing the LNCaP cells in continuous exposure of ENZ for three months in vitro . (B) Mice bearing LNCaP-ENZR xenografts were administered with LLU-206 (10, 25 mg/kg) via tail vein and tumor volumes were measured every other day ( n = 6). (C) Tumor weight of each group. (D) PSA levels of plasma were measured by ELISA kit. (E) Representative IHC staining of Ki-67. (F) Representative H&E staining of tumors. (G) The ARfl, ARv7, PSA protein levels in the tumors were detected by Western blot. (H) Representative bioluminescence images of 22RV1 xenografts in the prostate. (I) Tumor growth changes in mice bearing 22RV1 orthotropic tumors were analyzed by average fluorescence values ( n = 6). (J) The relative tumor weight was measured at the end of the experiment in each group. (K) Representative H&E staining of 22RV1 tumor tissues were presented as well as IHC analysis of Ki-67. (L) Ki-67 expression and tumor necrotic were quantified using Image J and Image-Pro Plus 6.0. (M, N) The ARfl, ARv7, and PSA protein levels in the tumors were detected by Western blot and quantified. Scale bars, 50 μm, 100 μm, 200 μm ∗ P < 0.05, ∗∗∗ P < 0.001. n.s., not significant.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: In Vivo, Generated, In Vitro, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Western Blot, Fluorescence, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: LLU-206 disrupts the ARfl/ARv7 heterodimers and the nuclear translocation. (A) The structure of ARfl and ARv7 comprises four functional domains: NTD (blue), DBD (red), hinge or CE3 (yellow) and the LBD (green). (B) A schematic diagram of the BiFC assay. Green fluorescence acquired with the dimerization of ARfl with ARfl or ARv7. (C) GFP fluorescence was determined by flow cytometry in PC3 cells cotransfected ARfl-C with ARfl-N or ARv7-N. (D) Co-IP assays with ARfl or ARv7 were performed using 22RV1 cells. The expression of ARfl and ARv7 was measured by Western blot. (E) The location of ARfl in LNCaP, treated with 5 μmol/L LLU-206 or ENZ for 24 h. (F) The location of ARfl and ARv7 in 22RV1 cells treated with LLU-206 or ENZ (5 μmol/L, 24 h, respectively). (G) The expression of ARfl protein levels in cytoplasm and nucleus of LNCaP cells treated with 10 μmol/L LLU-206 and ENZ for 24 h. (H) The expression of ARfl and ARv7 in cytoplasm and nucleus of 22RV1 cells treated with 10 μmol/L LLU-206 or ENZ for 24 h. Results are expressed as mean ± SEM, n = 3.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: Translocation Assay, Functional Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Flow Cytometry, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: LLU-206 suppresses ARfl and ARv7 transcriptional function/activity. (A) GSEA analysis of the RNA sequence for AR response, AR-induced and AR/ARv7 activated gene sets in 22RV1 cells treated with LLU-206 (10 μmol/L, 24 h) and control. ES, enrichment score. (B) Classical AR activated and repressed genes were shown in the RNA sequence data. (C) The mRNA levels of AR-activated or repressed genes were detected in LNCaP or LNCaP-ENZR cells treated with 10 μmol/L LLU-206 for 24 h ( n = 6). (D) The mRNA levels of AR and AR-activated gene were measured in LNCaP cells treated with 10 μmol/L LLU-206 for 24 h ( n = 6). (E, F) The mRNA levels of ARfl and ARv7 activated or repressed genes were detected in 22RV1 cells treated with LLU-206 for 24 h. (G) A luciferase assay was conducted in PC3 cells treated with LLU-206 with or without 10 nm R1881 for 24 h after transfection with ARfl and PSA-promoter luciferase plasmid. Results are expressed as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s., not significant.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: Activity Assay, Sequencing, Control, Luciferase, Transfection, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel inhibitor of ARfl and ARv7 induces protein degradation to overcome enzalutamide resistance in advanced prostate cancer

doi: 10.1016/j.apsb.2022.05.003

Figure Lengend Snippet: LLU-206 induces degradation through the MDM2-mediated ubiquitin–proteasomal system. (A) The protein levels of ARfl or ARv7 were detected in 22RV1 cells treated with LLU-206 (5 μmol/L, 10 μmol/L) and ENZ (10 μmol/L) for 48 h. (B) The effect of LLU-206 on ARfl and ARv7 stability was measured by Western blot. The protein levels were detected in 22RV1 cells, which were incubated with CHX (50 μg/mL) with or without LLU-206 for a different time. (C) Statistical analysis of the relative protein expression of ARfl and ARv7 for three independent experiments ( n = 3). (D) Western blot assay for the protein levels of ARfl and ARv7 in 22RV1 cells, which were treated with LLU-206 (10 μmol/L, 24 h), and MG132 (10 μmol/L, 6 h). (E) IP was conducted using anti-ARfl and immunoblotting was performed with ARfl, ARv7 and ubiquitin antibodies. (F) IP was conducted using anti-ARv7 and immunoblotting was performed with ubiquitin and ARv7 antibodies. (G) LLU-206 was docked with 63,890 human crystal structure proteins by computer modeling docking studies. The top 9 docking proteins are listed according to their binding energy score. (H) The docking model of LLU-206 (purple), AR-LBD (blue) and MDM2 (green). (I) Binding sites of LLU-206 with MDM2. (J) The ARfl, ARv7 and MDM2 protein levels were tested in 22RV1 cells with the treatment of MDM2 antagonist nutlin3 (5 μmol/L) and LLU-206 (10 μmol/L, 24 h). (K) Co-IP was conducted using anti-ARfl or anti-ARv7. Immunoblotting was performed with MDM2 and ARfl or ARv7 antibodies in 22RV1 cells.

Article Snippet: The specific experimental procedures were performed as previously described , with primary antibodies including ARfl (Abcam, Cat# ab74272), ARv7 (CST, Cat# 19672S), ARvs (Abcam, Cat# ab108341), PSA (CST, Cat# 5877S), Bax (Wanlei, Cat# WL01637A), Bcl-2 (Proteintech, Cat# 60178-1-lg), cleaved caspase 3 (Wanlei, Cat# WL03339a-020), MDM2 (CST, Cat# 86934S), Ubiquitin (Ub, Santa Cruz, Cat# sc-8017).

Techniques: Ubiquitin Proteomics, Western Blot, Incubation, Expressing, Binding Assay, Co-Immunoprecipitation Assay

Journal: Cancers

Article Title: Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.3390/cancers14235900

Figure Lengend Snippet: Effect of ACLY inhibition on the transcriptional activity of AR-FL and AR-V7. ( A ) Lysates from parental AR-negative and transformed PC-3 cells expressing the indicated constructs were subjected to Western blotting. ( B ) Parental PC-3 cells or PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK (Renilla) plasmids. The cells were cultured under androgen-depleted conditions (RPMI 1640 medium supplemented with 10% charcoal-stripped FBS) for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), enzalutamide (Enz) (2.5 μM), and the synthetic androgen agonist R1881 (1 nM) for 8 h. Samples were assayed for firefly and renilla luciferase activities using the Dual-Glo Luciferase assay. Values were normalized to Renilla activities. ( C ) PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK plasmids. The cells were cultured under androgen-depleted conditions for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), acetyl-CoA (0.1 mM), and R1881 (1 nM) for 8 h. Samples were assayed as described in B. Columns, means of three different experiments; bars, SDs. ** p < 0.001; *** p < 0.0001; ns—non-significant. Full size blots of are shown in .

Article Snippet: ORF of the AR was amplified with specific primers using EGFP-AR (plasmid #111215, Addgene, Watertown, MA, USA) as a template; ORF of the AR-V7 splice variant was amplified with specific primers using a AR-V7-pcw107 vector (plasmid #64635, Addgene, Watertown, MA, USA) as a template.

Techniques: Inhibition, Activity Assay, Transformation Assay, Expressing, Construct, Western Blot, Transfection, Cell Culture, Luciferase

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: Transcriptional re-activation of FKBP51 depends on AR-V7, not AR-FL in CRPC. (A) Western blot assays of AR-FL and AR-V7 expression in LNCaP-parental, LNCaP-Hormone Naïve P30 and LNCaP-AI cells. (B) Western blot detection of FKBP51 expression after AR-V7 and AR-FL overexpression in LNCaP-P30 cell, respectively. AR-FL, full length androgen receptor. (C) Western blot detection of FKBP51 expression in LNCaP-AI cell transfected with AR-V7 and AR-FL shRNA, respectively. AR-FL, full length androgen receptor. (D) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured without DHT, then, luciferase values were detected. (E) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured with DHT (10 nM), then, luciferase values were detected. (F) DNA pull down assays were used to determine the interaction of biotin-labeled FKBP51 promoter probe with AR-V7 proteins in LNCaP parental, LNCaP-P30 and LNCaP-AI cells. ***, P<0.001; NS, not significant. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Activation Assay, Western Blot, Expressing, Over Expression, Transfection, shRNA, Luciferase, Cell Culture, Labeling

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: AR-V7/FKBP51/NF-κB signaling axis promotes the progression of CRPC. (A) Western blot detection of indicated proteins in LNCaP-P30 cells transfected with indicated plasmids. GAPDH was used as a loading control. (B) Western blot detection of indicated proteins in LNCaP-AI cells transfected with indicated plasmids. GAPDH was used as a loading control. (C) Bioinformatic analysis of RNA-seq data from shAR-V7 and shAR-FL mediated knock-down experiments in LN95 cell lines. The RNA-seq data was retrieved fromGSE106560. Fold change of FKBP51 expression in response to AR-V7 knocked down (y axis), AR-FL knocked down (x axis) and DHT stimulation was presented. Size of green dot showed the concentration of DHT. The scales of x axis and y axis are different. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Western Blot, Transfection, Control, RNA Sequencing, Knockdown, Expressing, Concentration Assay

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: Level of AR-V7, FKBP51 and p-NF-κB are positively correlated in CRPC patients. (A) IHC staining of AR-V7, FKBP51, NF-κB, p-NF-κB p65 (Ser536) and c-Myc in the eleven CRPC specimens. For each sample, three different fields were randomly chosen and imaged under microscopy. Representative images were shown. (B,C) Quantitative analysis of IHC staining of the eleven CRPC tissues with Image J software. Average optical density = integrated density/area. CRPC, castration-resistant prostate cancer. The magnifications of these representative images are 10×, 20× and 40× in magnification order.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Immunohistochemistry, Microscopy, Software

Journal: Scientific Reports

Article Title: Constitutively Active AR-V7 Plays an Essential Role in the Development and Progression of Castration-Resistant Prostate Cancer

doi: 10.1038/srep07654

Figure Lengend Snippet: Comparison of patient characteristics according to AR-V7 expression

Article Snippet: Immunostaining of AR-V7 was performed using a mouse monoclonal anti-AR-V7 antibody (A&G Pharmaceutical, Columbia, MD, USA) and the Envision detection kit (Dako, Carpinteria, CA, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Constitutively Active AR-V7 Plays an Essential Role in the Development and Progression of Castration-Resistant Prostate Cancer

doi: 10.1038/srep07654

Figure Lengend Snippet: Cox regression analyses for time to CRPC in cohort 2 (104 metastatic PCa patients)

Article Snippet: Immunostaining of AR-V7 was performed using a mouse monoclonal anti-AR-V7 antibody (A&G Pharmaceutical, Columbia, MD, USA) and the Envision detection kit (Dako, Carpinteria, CA, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Constitutively Active AR-V7 Plays an Essential Role in the Development and Progression of Castration-Resistant Prostate Cancer

doi: 10.1038/srep07654

Figure Lengend Snippet: Cox regression analyses for cancer-specific survival after surgery in cohort 3 (46 CRPC patients)

Article Snippet: Immunostaining of AR-V7 was performed using a mouse monoclonal anti-AR-V7 antibody (A&G Pharmaceutical, Columbia, MD, USA) and the Envision detection kit (Dako, Carpinteria, CA, USA).

Techniques: Expressing