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: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells overexpressing USP12 was detected by ELISA. B The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells with USP12 knockdown was detected by ELISA. C , D Migration of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( C ) and USP12 knockdown ( D ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. E , F . Tube formation assay of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( E ) and USP12 knockdown ( F ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. The experiments were repeated three times. ** p < 0.01, *** p < 0.001; mean ± SD.

Article Snippet: Matrigel matrix (356234, Corning), the human VEGF-A ELISA kit (EKO588-96, Boster) and the human VEGF-C ELISA KIT (EKO539-96, Boster) were also used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression, Tube Formation Assay

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 3 The VEGF expression in tumor tissues. (A–C) VEGF staining in SGC-7901 observed with high microscope (200!original magnifications). (D–F) VEGF staining in MKN-45 observed with high microscope (200! original magnifications). A and D, control groups; B and E, low-dose rhGH groups; and C and F, high-dose rhGH groups. Three animals per group were used for immunohistochemistry assay. Full colour version of this figure available via http://dx.doi.org/10.1530/ JOE-11-0100.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Microscopy, Control, Immunohistochemistry

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 4 The mRNA expressions of angiogenesis-related factors in tumor tissues. (A) RT-PCR analysis of Ghr, Jak-2, Stat3, Vegf, Hif-1a, Fgf, and Mmp-2. Sc, control group of SGC-7901; Sl, low-rhGH treatment group of SGC-7901; Sh, high-rhGH treatment group of SGC-7901; Mc, control group of MKN-45; Ml, low-rhGH treatment group of MKN-45; Mh, high-rhGH treatment group of MKN-45. (B) Ratio of gray scale in SGC-7901 groups (*P!0.05 vs control and #P!0.05 vs low-dose rhGH group). (C) Ratio of gray scale in MKN-45 groups. Four animals per group were used for RT-PCR.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 5 The protein expressions of angiogenesis-related factors in tumor tissues. (A) Western blot analysis of STAT3, pSTAT3, VEGF, HIF-1a, and MMP-2. Sc, control group of SGC-7901; Sl, low-rhGH treatment group of SGC-7901; Sh, high-rhGH treatment group of SGC-7901; Mc, control group of MKN-45; Ml, low-rhGH treatment group of MKN-45; Mh, high-rhGH treatment group of MKN-45. (B) Ratio of gray scale in SGC-7901 groups (*P!0.05 vs control and #P!0.05 vs low-dose rhGH group). (C) Ratio of gray scale in MKN-45 groups. Four animals per group were used for western blotting.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Western Blot, Control