Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol selectively down-regulates VEGFR2 in ECs. a Western blots showing VEGFR2, VEGFR1, Tie2, FGFR1, and β-actin expression in HUVECs after 4-hour treatment with 0, 2.5, 5, or 10 µM clioquinol. b - e Expression level (% of 0 µM) of VEGFR2 ( b ), VEGFR1 ( c ), Tie2 ( d ), and FGFR1 ( e ) normalized to β-actin in HUVECs treated as described in (a) ( n = 3 independent experiments). f Mean fluorescence intensity (MFI) of membrane VEGFR2 on HUVECs treated with or without 10 µM clioquinol for 0.5, 1, 2, 3, and 4 h, as assessed by flow cytometry ( n = 4). g Phase-contrast microscopic images of HUVEC spheroids after 24-hour treatment without or with clioquinol in the absence or presence of 25 ng/mL VEGF. Scale bar: 135 μm. h Sprouting (% of control) of HUVEC spheroids treated as described in (g) ( n = 13–15). i Western blots showing VEGFR2 and β-actin expression in HUVECs, HDMECs, hPC-PLs, NHDFs, MCF-7, MDA-MB-231, and 4T1-Luc2 cells. j Expression level (% of HUVEC) of VEGFR2 normalized to β-actin in different cell types as described in (i) ( n = 3 independent experiments). k Correlation between cell viability and VEGFR2 expression following exposure to 10 or 25 µM clioquinol. Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (b-e, h, j: one-way ANOVA with Tukey’s multiple comparisons test; f: unpaired Student’s t-test; k: Pearson correlation coefficient)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Western Blot, Expressing, Fluorescence, Membrane, Flow Cytometry, Control

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol binds to the ATP-binding pocket of VEGFR2 and causes its degradation. a Western blots showing VEGFR2 and β-actin expression in HUVECs treated with 0.1% DMSO (vehicle) or 10 µM clioquinol in the presence of 100 µM cycloheximide (CHX) for 0, 1, 2, 4, 6, and 8 h. b Expression level (% of 0 h) of VEGFR2 normalized to β-actin in HUVECs treated as described in ( a ) ( n = 3 independent experiments). c mRNA level of VEGFR2 (% of control) in HUVECs treated with 0.1% DMSO (control) or 10 µM clioquinol for 4 h, as assessed by real-time PCR ( n = 3). d Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated without or with 20 µM MG132 or 200 µM chloroquine (CQ) for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. e Expression level (% of control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (d) ( n = 3 independent experiments). f Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated with 4 µg/mL IgG, 4 µg/mL anti-VEGFR2 NAb, 0.1% DMSO (vehicle), 100 nM lenvatinib, 250 nM tivozanib, or 1 mM ATP for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. g , h Expression level (% of IgG or control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (f) ( n = 4 independent experiments). i Western blots showing p-VEGFR2, VEGFR2, and β-actin expression in HUVECs that were treated with 0.1% DMSO, 100 nM lenvatinib, 250 nM tivozanib or 10 µM clioquinol for 1 h and then stimulated with 25 ng/mL VEGF for 7 min. j Expression level (% of control) of p-VEGFR2 normalized to VEGFR2 in HUVECs treated as described in (i) ( n = 4 independent experiments). k VEGFR2 kinase activity (% of control) in the presence of serial dilutions of clioquinol at an ATP concentration of 10 µM, as assessed by VEGFR2 kinase assay ( n = 2). l VEGFR2 kinase activity (% of control) in the presence of lenvatinib (0.3, 1, and 3 nM) or clioquinol (10, 50, and 250 µM) at ATP concentrations of 10 or 500 µM, as assessed by VEGFR2 kinase assay ( n = 3). Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (e, g, h, j: one-way ANOVA with Tukey’s multiple comparisons test; b, c, l: unpaired Student’s t-test)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Binding Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Activity Assay, Concentration Assay, Kinase Assay

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol and MK-2206 inhibit TNBC development, as assessed by histology and immunohistochemistry. a Light microscopic images of H&E-stained 4T1 tumors (bordered by dotted line) in control, clioquinol, MK-2206, and combination group. Scale bar: 160 μm. b Tumor size (mm 2 ) in control, clioquinol, MK-2206, and combination group, as assessed by histology ( n = 10). c Fluorescence microscopic images of tumor microvessels in control, clioquinol, MK-2206, and combination group. Tumor sections were stained with an anti-CD31 antibody (red) and Hoechst 33342 (blue) for the visualization of ECs and cell nuclei, respectively. Scale bar: 70 μm. d Microvessel density (mm − 2 ) of 4T1 tumors in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry ( n = 10). e , g Light microscopic images of Ki67- (e) and cleaved caspase-3-positive (g) tumor cells in control, clioquinol, MK-2206, and combination group. Scale bars: 55 μm. f , h Ki67- (f) and cleaved caspase-3-positive tumor cells (h) (% of total cell number) in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry ( n = 10). i Fluorescence microscopic images of tumor microvessels in control and clioquinol group. Tumor sections were stained with an anti-VEGFR2 antibody (red), an anti-CD31 antibody (green), and Hoechst 33342 (blue). Scale bar: 60 μm. j Area of VEGFR2 signal normalized to CD31 area (% of control) in tumors of control and clioquinol group. (k) VEGFR2 MFI (% of control) in tumors of control and clioquinol group. Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (b, d, f, h: one-way ANOVA with Tukey’s multiple comparisons test; j, k: unpaired Student’s t-test)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Immunohistochemistry, Staining, Control, Fluorescence

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Scheme illustrating the molecular mechanisms underlying the potent inhibitory effects of clioquinol alone and its synergistic inhibitory effects with MK-2206 on angiogenesis. Clioquinol binds directly to the ATP-binding site of VEGFR2 on ECs, leading to a transient inhibition of VEGFR2 phosphorylation induced by VEGF and eventual promotion of VEGFR2 degradation via both the proteasome and lysosome systems. Consequently, the downstream ERK pathway is down-regulated. Furthermore, clioquinol increases AKT phosphorylation, while the inhibition of AKT by MK-2206 synergistically enhances the anti-angiogenic efficacy of clioquinol

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Binding Assay, Inhibition, Phospho-proteomics