Journal: Molecular Cancer Therapeutics

Article Title: Nifuroxazide Activates the Parthanatos to Overcome TMPRSS2:ERG Fusion-Positive Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0159

Figure Lengend Snippet: RNA-seq analysis of VCaP cells treated with NFZ. A, Differential gene enrichment map. On the left is a pathway enrichment map of differential genes. Red font represents upregulation, while green represents downregulation. On the right is a heatmap of the differential gene expression between untreated and NFZ-treated VCaP cells. Results are shown as three independently repeated groups. NFZ groups were treated with 1 μmol/L NFZ for 24 hours. B, Influence of NFZ on DNA repair pathway. C, Effect of NFZ on the cell cycle. VCaP cells were treated with 5, 10, or 20 μmol/L NFZ for 24 hours. Results are shown as mean ± SD ( n = 3 independent experiments with at least 10,000 cells counted in each replicate; ns, not significant; **, P < 0.01; ***, P < 0.001), compared with DMSO group by Dunnett multiple comparisons test.

Article Snippet: Xenograft tumors were established by injecting 4 × 10 6 VCaP cells subcutaneously on the left abdomen of male SCID mice, obtained from Charles River Laboratories.

Techniques: RNA Sequencing, Gene Expression

Journal: Molecular Cancer Therapeutics

Article Title: Nifuroxazide Activates the Parthanatos to Overcome TMPRSS2:ERG Fusion-Positive Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0159

Figure Lengend Snippet: NFZ activates parthanatos by blocking the interaction of ERG and PARP1. A, The combined effect of OLP and NFZ on VCaP cells. VCaP cells were treated with 5 μmol/L NFZ or 5 μmol/L NFZ combined with 0.5, 1.0, or 20 μmol/L OLP. The results in the histogram are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001), compared with the DMSO group by Dunnett multiple comparisons test. B, Co-immunoprecipitation of ERG and PARP1 from ERG-overexpressing cells (DU145-ERG). Representative Western blotting is shown on top, and a quantitative histogram is shown beneath. Histogram results are shown as mean ± SD ( n = 5 independent experiments; ns, not significant; *, P < 0.05; **, P < 0.01), compared with the DMSO group by Dunnett's multiple comparisons test. C, Illustration of parthanatos activated by NFZ. NFZ reduces the interaction of ERG and PARP1, so that overactive PARP1 activates the parthanatos pathway. D, RT-qPCR for AIF mRNA on VCaP cells. RQ, relative quantification. Abscissa indicates 2 or 5 μmol/L NFZ treatment for 12 or 24 hours. E, AIF Western blot. F, γ-H2A.X Western blotting. Representative Western blotting is shown on top, and a quantitative histogram is shown beneath it for the diagrams in E and F. VCaP cells were treated with DMSO, or 2, or 5 μmol/L NFZ, or NFZ combined with 10 μmol/L OLP for different times. Intensity was measured using Image lab 5.2 software and normalized to total protein. Relative expression is shown using the first sample as a reference. Uncropped blots are shown in Supplementary Figs. S6, 8S and 9S. Histogram results are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05;**, P < 0.01; ***, P < 0.001; ****, P < 0.0001), compared with the DMSO group by Dunnett multiple comparisons test. G, IC 50 values for ERG-overexpressing cells. The DU145 vector group is the negative control group. The DU145-ERG group is the ERG-overexpressing group, which was treated with 0.1, 0.5, 1, 5, 10, and 30 μmol/L NFZ, or a mixture of the NFZ and 10 μmol/L OLP. IC 50 values were obtained using GraphPad Prism 8.0. OLP, olaparib.

Article Snippet: Xenograft tumors were established by injecting 4 × 10 6 VCaP cells subcutaneously on the left abdomen of male SCID mice, obtained from Charles River Laboratories.

Techniques: Blocking Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Quantitative Proteomics, Software, Expressing, Plasmid Preparation, Negative Control

Journal: Molecular Cancer Therapeutics

Article Title: Nifuroxazide Activates the Parthanatos to Overcome TMPRSS2:ERG Fusion-Positive Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0159

Figure Lengend Snippet: NFZ induces apoptosis and necrosis. A, Western blotting for cleaved PARP1. Representative Western blot is shown on top and the quantitation is shown beneath. VCaP cells were treated with DMSO, or 2, or 5 μmol/L NFZ, or NFZ combined with 10 μmol/L OLP for different times. Band intensity was measured by Image Lab 5.2 software and normalized to total protein. Relative levels are expressed using the first sample as a reference. Uncropped blots are shown in Supplementary Fig. S10. Results in the histogram are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05;**, P < 0.01; ***, P < 0.001; ****, P < 0.0001), compared with the DMSO group by Dunnett multiple comparisons test. B, Confocal laser imaging for cleaved PARP1 and AIF. VCaP cells were treated with 2 μmol/L NFZ for 30 minutes. Immunofluorescence of cleaved PARP1 (green), AIF (red) and nuclei (DAPI, blue). Scale bars, 5 μm. C, Confocal laser imaging for γ-H2A.X. VCaP cells were treated with 2 μmol/L NFZ for 12 hours. Immunofluorescence of γ-H2A.X (green) and nuclei (DAPI, blue). Scale bars, 5 μm. D, Flow cytometry for cell death. Apoptotic cells and dead cells are stained by Hoechst to different degrees. VCaP cells were treated with 5 μmol/L NFZ, 10 μmol/L NFZ, or 20 μmol/L NFZ for 48 hours or 72 hours. Q1 and Q2 gate are the necrotic cells; Q3 and Q4 represent the normal cells and apoptotic cells, respectively. The results show the proportion of dead cells to the total number with at least 10,000 cells counted. DMSO was used as a control. OLP, olaparib.

Article Snippet: Xenograft tumors were established by injecting 4 × 10 6 VCaP cells subcutaneously on the left abdomen of male SCID mice, obtained from Charles River Laboratories.

Techniques: Western Blot, Quantitation Assay, Software, Imaging, Immunofluorescence, Flow Cytometry, Staining, Control

Journal: iScience

Article Title: Leveraging Vγ9Vδ2 T cells against prostate cancer through a VHH-based PSMA-Vδ2 bispecific T cell engager

doi: 10.1016/j.isci.2024.111289

Figure Lengend Snippet: Target binding, Vγ9Vδ2 T cell activation, and tumor cell lysis by the PSMA-Vδ2-Fc bsTCE (A) PSMA-Vδ2-Fc bsTCE and control bsTCE binding to expanded Vγ9Vδ2 T cells ( n = 6), LNCaP (left: n = 3, right: n = 2), and PC3 cells ( n = 1). (B) PSMA expression by 22Rv1 and VCaP shown as histograms. (C) Vγ9Vδ2 T cell CD107a expression, CD25 expression, and IFN-γ secretion ( n = 3). (D) Target cell lysis ( n = 3–4) in 24 h co-cultures of expanded Vγ9Vδ2 T cells with 22Rv1, VCaP, LNCaP (WT or PSMA KO), or PC3 with indicated concentrations of bsTCE. (E) Vγ9Vδ2 T cell CD25 expression ( n = 3) and target cell lysis ( n = 3) in 24 h co-cultures of expanded Vγ9Vδ2 T cells with 22Rv1, VCaP, LNCaP, or PC3 with 100 nM PSMA-Vδ2-Fc bsTCE or control bsTCEs. Data were generated using flow cytometry (A left, B, C; CD107a and CD25, D, E), ELISA (A, middle and right) or CBA (C, IFNγ). Data represent mean ± S.E.M.

Article Snippet: LNCaP (PSMA + clone FGC, CRL-1740) and PC3 (PSMA − , CRL-1435) were obtained from American Type Culture Collection (ATCC) and VCaP (PSMA + , 06020201-1VL) and 22Rv1 (PSMA + , 5092802) from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Binding Assay, Activation Assay, Lysis, Control, Expressing, Generated, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Leveraging Vγ9Vδ2 T cells against prostate cancer through a VHH-based PSMA-Vδ2 bispecific T cell engager

doi: 10.1016/j.isci.2024.111289

Figure Lengend Snippet: Impact of DNAM-1 and NKG2D receptor interactions in PSMA-Vδ2-Fc bsTCE mediated Vγ9Vδ2 T cell activation and prostate tumor cell lysis (A) CD107a expression (4 h culture, n = 4) and (B) tumor cell lysis (24 h culture, n = 4) from cultures of expanded Vγ9Vδ2 T cells pre-incubated with 10 μg/mL anti-Fc receptor Ab and α-DNAM-1 blocking antibody or IgG1 isotype control with LNCaP, VCaP, or 22Rv1 tumor cells with indicated concentrations of PSMA-Vδ2-Fc bsTCE. (C and D) (C) CD107a expression (4 h culture, n = 4) and (D) tumor cell lysis (24 h culture, n = 4) from cultures of expanded Vγ9Vδ2 T cells pre-incubated with 10 μg/mL anti-Fc receptor Ab and α-NKG2D blocking antibody or IgG1 isotype control with LNCaP, VCaP, or 22Rv1 tumor cells with indicated concentrations of PSMA-Vδ2-Fc bsTCE. (E) CD107a expression on Vγ9Vδ2 T cells derived from malignant prostate tissue ( n = 3) of PCa patients after 4 h co-culture with 0.05 nM PSMA-Vδ2-Fc bsTCE and α-DNAM-1 or α-NKG2D blocking Ab. Data were all generated using flow cytometry and circles represent individual data-points. Box and whisker plots indicate the median, 25th to 75th percentiles and minimum to maximum and bar graphs represent mean ± S.E.M. Paired t test (A, C–E). p < 0.05: ∗, p < 0.01: ∗∗ and p < 0.001: ∗∗∗.

Article Snippet: LNCaP (PSMA + clone FGC, CRL-1740) and PC3 (PSMA − , CRL-1435) were obtained from American Type Culture Collection (ATCC) and VCaP (PSMA + , 06020201-1VL) and 22Rv1 (PSMA + , 5092802) from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Activation Assay, Lysis, Expressing, Incubation, Blocking Assay, Control, Derivative Assay, Co-Culture Assay, Generated, Flow Cytometry, Whisker Assay