Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in VEGF-stimulated HUVECs. Cells were serum-starved for 6 h and then pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by stimulation with VEGF (100 ng/mL) for another 10 min (VEGFR2, PLCγ1, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before proteins were collected. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using respective antibodies. ( A ) E7050 inhibited the phosphorylation (Tyr1175) of VEGFR2 induced by VEGF in HUVECs. ( B ) The quantified results show that the ratio of p-VEGFR2 protein normalized to the total amount of VEGFR2 protein, which was measured by densitometry. ( C ) E7050 inhibited the phosphorylation of PLCγ1, FAK, and Src in VEGF-stimulated HUVECs. Calculated ratios of ( D ) p-PLCγ1, ( E ) p-FAK, and ( F ) p-Src normalized to the relative total protein levels are shown. ( G ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. The compiled results of the ratios of ( H ) p-Akt, ( I ) p-JNK, and ( J ) p-p38 MAPK normalized to the relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effect of E7050 on the expression level of HGF in MES-SA/Dx5 cells. ( A ) Cells were treated with various concentrations (5–25 μM) of E7050 for 24 h. Whole cell extracts were prepared and subjected to Western blotting using antibodies against HGF and β-actin. β-actin was used as an internal loading control. ( B ) Densitometric analysis of blots relative to HGF protein after normalization with β-actin. ( C ) Secreted HGF in cell culture media was determined by ELISA. Data are presented as the mean ± SEM of three independent experiments. * p < 0.05 versus vehicle-treated control cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in cultured MES-SA/Dx5 cells-derived conditioned medium (CM)-treated HUVECs. Cells were serum-starved for 6 h and pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by the addition of VEGF (100 ng/mL) or CM (from cultured MES-SA/Dx5 cells) for another 10 min (VEGFR2, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before protein extraction. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using specific antibodies. ( A ) E7050 inhibited the phosphorylation of VEGFR2 and Src in MES-SA/Dx5 CM-induced HUVECs. ( B ) The relative band density of p-VEGFR2 protein was normalized to total VEGFR2 protein, which was measured by densitometry. Calculated ratios of ( C ) p-FAK and ( D ) p-Src normalized to the relative total protein levels are shown. ( E ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in MES-SA/Dx5 CM-induced HUVECs. The compiled results of the ratios of ( F ) p-Akt, ( G ) p-JNK, and ( H ) p-p38 MAPK normalized to relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Cell Culture, Derivative Assay, Protein Extraction, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on tumor growth and angiogenesis in the MES-SA/Dx5 cell line-derived xenograft mouse model. Histological characteristics in the tumor tissue sections of MES-SA/Dx5 xenografts obtained from vehicle- and E7050-treated nude mice on day 28 were measured by H&E staining. The expression levels of CD31, VEGF, and p-VEGFR2 (Tyr1175) in tumor tissue sections were also examined by immunohistochemical analyses. Representative photomicrographs of H&E and immunohistochemical staining in tumor tissue sections from vehicle control and E7050-treated groups of mice are shown. Scale bar: 100 μm.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Derivative Assay, Staining, Expressing, Immunohistochemical staining, Control

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Schematic diagram of a proposed mechanism of E7050-induced anti-angiogenic activity. E7050 exerts anti-angiogenic effects in VEGF-stimulated endothelial cells by downregulating the phosphorylation of VEGFR2 and its downstream mediators, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK. The blockage of VEGFR2-mediated signaling cascade pathways by E7050 contributes to the inhibition of proliferation, migration, and tube formation in endothelial cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Activity Assay, Phospho-proteomics, Inhibition, Migration

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Binding activities of BsAb: ( A ) Surface plasmon resonance sensor grams showing the binding kinetics of anti-EGFR/VEGFR2 BsAb to antigens EGFR and VEGFR2 as detected by a Biacore T200 optical biosensor instrument. Black line represents the constant concentration of anti-EGFR/VEGFR2 BsAb (8 µg/mL) and colored lines represent the nM concentrations of antigens (EGFR or VEGFR2). ( B ) ELISA binding assay showing that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 comparable with cetuximab and ramucirumab, respectively. ( C ) Flow cytometry experiment was performed to assess the binding of anti-EGFR/VEGFR2 BsAb, cetuximab, ramucirumab with cell surface EGFR and VEGFR2 expressed on MDA-MB-231, BT-20, MDA-MB-468 and HUVEC cells. Human IgG was used as isotype control. ( D ) Whole cell lysate of MDA-MB-231 cells was subjected to co-immunoprecipitation assay to assess the binding of anti-EGFR/VEGFR2 BsAb with EGFR and VEGFR2 comparable to parental antibodies. Unprocessed western blot images are available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Binding Assay, SPR Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Anti-EGFR/VEGFR2 BsAb inhibited ligand-induced activation of EGFR and VEGFR2. ( A ) MDA-MB-231 cells were serum starved overnight and then pre-treated with different antibodies at concentration of 10 µg/mL for 1 h followed by a co-treatment of EGF and VEGF-A (EGF + VEGF) at a concentration of 50 ng/mL each for 30 min. After treatments, WCL was prepared and subjected to Western blot analysis to determine the phosphorylation of EGFR. ( B ) MDA-MB-231 cells were treated as described in A and then phosphorylation levels of Akt was determined by western blot analysis. ( C ) BT-20 cells were exposed to the treatment conditions as described in the A and analyzed by western blotting to assess the phosphorylated and total levels of EGFR, ERK and AKT. ( D ) HUVEC cells were treated with antibodies and EGF + VEGF as described in A. After treatments, WCL was analyzed by western blot to determine the phosphorylated and total levels of VEGFR2, ERK, Akt using respective antibodies. ( E ) MDA-MB-231 cells were serum starved overnight in media containing 1% FBS and then treated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 or left untreated. Western blotting was carried out to evaluate the expression of total VEGFR2. Unprocessed western blot images and available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Activation Assay, Concentration Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Anti-EGFR/VEGFR2 BsAb downregulates cancer cell-induced VEGFR2 signaling in HUVEC cells ( A ) The levels of VEGF-A were determined by ELISA assay in supernatants of MDA-MB-231, MDA-MB-468 and BT-20 cells after culturing the cells in a 6-well-plate for 2 days at 37 °C. ( B ) MDA-MB-231 cells were cultured for 2 days in serum free media and then conditioned media was harvested. HUVEC cells were incubated for 30 min and 2 h with the conditioned media obtained from MDA-MB-231. After incubation, Western blot analysis was performed to assess the phosphorylated and total levels of VEGFR2 and ERK. ( C ) HUVEC cells were pretreated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 BsAb or left untreated and then incubated with conditioned media obtained from MDA-MB-231 cells. Post-incubation, western blotting was performed to assess the phosphorylated and total levels of VEGFR2 and ERK. ( D ) HUVEC cells were cultured in the top chamber of trans-well co-culture plates and MDAMB231 cells were cultured in a separate 6-well plate in serum free media overnight. Both HUVEC and MDAMB231 cells were then treated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 BsAb for 1 h or left untreated. After treatments, the trans-well upper chamber inserts containing HUVEC were placed on top of 6-well plate containing MDA-MB-231 cells for 30 min. WCL of HUVEC cells was collected and subjected to Western blot analysis to evaluate the phosphorylated levels of ERK and VEGFR2. ( E ) HUVEC cells and MDA-MB-231 cells were cultured in trans-well co-culture plate as described in D and treated with 1, 5 and 20 µg/mL of anti-EGFR/VEGFR2 BsAb. WCL of HUVEC cells was then subjected to Western blot analysis to assess the levels of ERK and VEGFR2. Unprocessed western blot images are available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Western Blot, Co-Culture Assay