Figure 5.

Anti-angiogenesis and anti-proliferation functions of cm. from cancer cells on HUVECs through paracrine stimulation. (A) VEGF levels in ART/NVB treated cancer cells and cmART/NVB treated HUVECs (B) Quantification of the effects of cm. from MDA-MB-231 cells on angiogenesis by performing capillary-like tube-formation, wound-healing, and migration assays. HUVECs were cultured with cellular VEGF protein derived from a cell lysate and with VEGF protein secreted into cm. by cancer cells treated with or without ART or/and NVB. (C) Western blot analysis of MMP2, MMP9, VEGFA, and p-KDR expression in HUVECs after cmART/NVB exposure. (D) These cell populations were subsequently treated with cmART/NVB. Cellular DNA synthesis was measured by EdU-incorporation assays and flow cytometry. The percentage of S phase cells is indicated. (E) Images showing a rat aorta ring incubated for 7 days with the indicated cm. Representative images of a rat aortic ring sprouting in response to the indicated treatments. Quantitative sprout growth data are shown. Scare bar = 200 μm. (F) The chorioallantoic membrane of a 7-day-old chick embryo onto which a single small ring was placed. These rings were treated with cm. from Ctrl or ART/NVB-treated cells for 48 h. (G) Five-week-old SCID mice were injected with 0.5 mL of Matrigel mixed with cmART/NVB. Matrigel was injected as the vehicle control. After 14 days, the skin of the sacrificed experimental animals was pulled back to expose the intact Matrigel plugs, which were analyzed. Scare bar = 0.5 mm. (H) Inhibition of blood vessel formation by cmART/NVB. The Matrigel plugs were fixed, sectioned, and stained with an anti-CD31 antibody and counterstained with hematoxylin and eosin (H&E; magnification, ×200). (I) Western blot analysis of the relative protein-expression levels of VEGF and downstream signaling molecules following cmART/NVB treatment in HUVECs. The cellular expression levels observed in the absence of drug treatment were normalized as 1.0. *P < 0.05, **P < 0.01, and ***P < 0.001