Table 1.
IC50 values for aeroplysinin-1 treatment on human endothelial cells determined by the MTT assay.
Figure 1.
Aeroplysinin-1 inhibits angiogenesis in human endothelial cells.
A) Inhibition of the formation of "tubule-like" structures of human immortalized endothelial cells on Matrigel. Representative photographs of control (untreated), and aeroplysinin-1-treated human immortalized endothelial cells on Matrigel after 6 h of treatment. Data are shown on the minimum aeroplysinin-1 concentration yielding total inhibition in each cell line. The histogram shows quantitative analysis of data for the full range of tested endothelial cell types and compound concentrations. B) Aeroplysinin-1 inhibits the expression levels of MMP-2 detected in gelatinolytic assays. The figure corresponds to representative results of gelatinolytic assays showing the levels of MMP-2 activity in control (untreated), and aeroplysinin-1-treated human immortalized endothelial cell conditioned media. C) Aeroplysinin-1 treatment has only a weak inhibitory effect on RF-24 immortalized HUVEC migration in a "wound healing" assay (histogram, above). This effect could not be detected in a semicontinuous fluorescence assay of migration (time course curves, below). In this last case, cells were preloaded with a fluorescence marker and the migration assay was carried out as described in Material and methods. Data are fluorescence measurements of the migrating cells into the lower well at different times of incubation. D) Aeroplysinin-1 treatment has no relevant effect on RF-24 immortalized HUVEC in an invasion assay. Cells were preloaded with a fluorescence marker and the invasion assay was carried out as described in Material and methods. Data are fluorescence measurements of the “invading” cells into the lower well at different times of incubation. All the experiments were carried out as described in Material and methods. Data represent mean±SD for three independent experiments. Symbols indicate significant differences between control-untreated and treated cells (*p<0.01).
Figure 2.
Aeroplysinin-1 decreases the expression levels of MCP-1 and TSP-1 in HUVEC.
A) A typical result with GE-Array Q Series Human Angiogenesis Gene Array (SuperArray) is shown. B and C) Validation of the effects of aeroplysinin-1 on the expression levels of MCP-1. MCP-1 messengers were detected by semi-quantitative RT-PCR, using the levels of amplification of GAPDH as a control (B). MCP-1 protein was quantitatively determined by using an ELISA (C). D) Validation of the effects of aeroplysinin-1 on the expression levels of TSP-1. TSP-1 protein levels were detected by Western blotting, using the levels of β-actin as a control. Experiments were carried out as described in the Experimental Section.
Figure 3.
Aeroplysinin-1 decreases the expression levels of MCP-1, TSP-1, ETL, IL-1, FADD and MMP-1, key pro-inflammatory biomolecules in HUVEC.
A) A typical result with Human Antibody L-series 507 Cytokine Arrays (RayBiotech) is shown. B) Validation of the effects of aeroplysinin-1 on the expression levels of ETL, IL-1α, FADD and MMP-1. Protein expression levels were detected by Western blotting. C) Quantification of protein levels in aeroplysinin-1-treated samples detected by Western blotting, using the levels of β-actin as a control. Experiments were carried out as described in the Experimental Section. Data are given as percentages of expression, taking the correspondent values of the respective controls as 100%. Means±S.D. for three independent experiments are provided.
Figure 4.
Aeroplysinin-1 abrogates the expression of PMA-induced COX-2 protein.
A) PMA (50 ng/mL)-induced COX-2 mRNA was detected by qPCR, using the levels of amplification of GAPDH as a control. B) PMA (50 ng/mL)-induced COX-2 protein levels were detected by Western blotting as described, using the levels of β-actin as a control. C) The expression levels of the protein COX-2 were also analyzed in experiments carried out in the presence of cycloheximide (CHX), as shown in a Western blot. Cells were treated with cycloheximide (90 µg/mL) for 1 h. After washing both control and CHX-pretreated cells were treated in complete medium with PMA (50 ng/mL) in the presence or absence of aeroplysinin-1 (10 µM) for 4.5 h. D) The histogram shows the quantification of three independent experiments as that shown in C.
Figure 5.
Aeroplysinin-1 inhibits key processes and the expression levels of key biomolecules in THP-1 human inflammatory monocytes.
A) Aeroplysinin-1 inhibits THP-1 proliferation as determined by the MTT method. B) Aeroplysinin-1 significantly decreases the expression levels of MCP-1 and COX-2 mRNA as determined by qPCR. C) Aeroplysinin-1 significantly decreases the expression levels of COX-2 protein as determined by Western blot. D) Aeroplysinin-1 has no effect on THP-1 migration. E) Aeroplysinin-1 has no effect on THP-1 invasion. Experiments were carried out as described in Material and methods. Data represent mean±SD for three independent experiments (each one with four replicates of each tested concentration).
Table 2.
Primers, amplicon sizes and qPCR conditions.