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Fig. 1. LYR analogs inhibit tube formation of HUVECs. (A) LYR analogs (LYR-1 to LYR-8) are structurally related to classical cannabinoid Δ9-tetrahydrocannabinol (THC). Chemical structure of Δ9-THC is shown in the box. (B) HUVECs (1 × 104) were plated in a well coated with Matrigel basement membrane matrix. Cells were treated with 5 μM LYR analogs in the presence of VEGF (20 ng/ml). After 14 h, cells were photographed with a digital camera under a phase contrast microscope at 100× magnification. (C) Experiment was carried out as described above with the indicated concentrations of LYR-7 or LYR-8 for 14 h. (D) Total tube length per field was measured by ImageInside software. The bar graph shows the means ± S.E.M. of the experiment carried out in triplicate. *P < 0.05, compared to VEGF-stimulated control group.

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Fig. 2. LYR-7 and LYR-8 inhibit migration and invasion of HUVECs. (A) Scratch wound migration of HUVECs: inhibitory effect of LYR-7 and LYR-8 on VEGF-induced HUVEC migration. Inactivated HUVECs were subjected to wound-healing migration assays and the migrating cells were counted using ImageInside software. Experiment was performed in triplicate. (B) Chemotactic migration in Transwell: effect of LYR analogs on VEGF-induced HUVEC migration in the Transwell assay. Red cells with irregular shape were migrating cells attached on the outside surface of the top chamber. (A–B) The bar graphs in the right panel show the summary of quantitative results of the average number of migrating HUVECs ± S.E.M. *P < 0.05, compared to VEGF-stimulated control group.

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Fig. 3. Inhibitory effects of LYR-7 and LYR-8 on growth factor-induced cell proliferation and TNF-α-induced NF-κB transcriptional activity. (A) HUVECs (1 × 104 per well in a 48 well-plate) were starved with 0.2% FBS medium and then treated with VEGF (20 ng/ml) and different concentrations of LYR analogs for 48 h. Cell viability was quantified by MTT assay. Values are means ± S.E.M. of eight measurements. *P < 0.05 versus VEGF alone. (B) HUVECs were cultured and stimulated with VEGF as described above. AM281 (1 μM) and AM630 (1 μM) were pre-treated for 1 h before the LYRs (5 μM) and VEGF co-treatment. Cell viability was quantified by MTT assay. Values are means ± S.E.M. of eight measurements. #P < 0.05 versus vehicle-treated control. *P < 0.05 versus VEGF alone. (C–D) The effects of LYR analogs on serum-treated proliferation of HUVECs, MCF-7 and TAMR-MCF-7 cells: The treatment is the same as in A and B. The data points represent three experiments performed in triplicate. (E) NF-κB-luc and pRL-TK were introduced into HT29 cells using GeneJammer reagent. Cells were then pre-treated with or without LYR analogs (5 μM) for 1 h, followed by TNF-α (10 ng/ml) for 3 h. Luciferase activity was measured and expressed as relative luciferase units (RLU) (firefly luciferase/Renilla luciferase). Bar graphs show the means ± S.E.M. of three experiments. #P < 0.05 versus vehicle-treated control. *P < 0.05 versus TNF-α-stimulated group.

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Fig. 4. LYR-7 and LYR-8 inhibit the VEGF-induced angiogenesis in vivo. (A) VEGF (20 ng/CAM) or vehicle (0.1% BSA in PBS) was loaded onto a dried cortisone-saturated filter disk placed on an avascular area of the CAM. The disks were treated with various concentrations of LYR-7 or LYR-8. After 72 h incubation, the CAM membrane was resected and imaged under microscope. (B) Quantitation of new branches formed from existing blood vessels: Photographs were imported into imaging software to quantitate the number of new branches formed. The bar graph represents the mean number of branch points ± S.E.M. of at least six chick embryos. #P < 0.05, compared to PBS-treated control. *P < 0.05, compared to VEGF-treated group.

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Fig. 5. LYR-7 and LYR-8 inhibit VEGF expression in TAMR-MCF-7 cells. (A) TAMR-MCF-7 cells were treated with the indicated concentrations of LYR analogs for 18 h, and supernatants were collected. The secreted level of VEGF was quantified by ELISA as described in Materials and methods. The bar graph represents the means ± S.E.M. of six analyses. *P < 0.05, compared to vehicle-treated control. (B) The TAMR-MCF-7 cells were treated as above and whole cell lysates were used to determine cellular VEGF levels by western blot analysis. (C) The band densities were measured and presented in a graph showing relative density ± S.E.M. of three independent experiments. *P < 0.05, compared to vehicle-treated control.

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Fig. 6. LYR-7 and LYR-8 inhibit cancer-induced angiogenesis and tumor growth. (A) For tumor implantation on CAM, 1.5 × 106 TAMR-MCF-7 cells were loaded onto each CAM and a single dose (20 μM) of LYR-7 or LYR-8 was given at the time of implantation. After 5 days of incubation, the CAM tissues were resected and digital images were captured. (B) A parallel experiment was carried out with MCF-7 cells. As shown in the photographs, LYR-7 and LYR-8 inhibited both angiogenesis and tumor growth (size). (C) New blood vessels were quantified and expressed as percentage of PBS-treated control. #P < 0.05, compared to PBS- or VEGF-treated groups. $P < 0.05 versus vehicle-treated MCF-7 cell-implanted group. *P < 0.05, compared to vehicle-treated cancer cell (TAMR-MCF-7 or MCF-7)-implanted group.

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1
Current address: College of Pharmacy, Hanyang University, 1271, Sa-3-Dong, Ansan 426-791, South Korea.

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