Lonidamine causes inhibition of angiogenesis-related endothelial cell functions

Abstract

The aim of this study was to assess whether lonidamine (LND) interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 microg/ml). In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 microg/ml), whereas 50 microg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.

Figure 1

Low doses of LND inhibit endothelial cell proliferation while not inducing apoptosis. (A) HUVEC proliferation was evaluated after exposure for 48 hours to doses of LND ranging from 0.1 to 50 g/ml (black column). (B) Cell proliferation of HMVEC (dark grey column), EA.hy926 (pale grey column), and BAEC (white column) was evaluated after exposure for 48 hours to 10 g/ml LND. Cells exposed to medium with or without 10% FCS were used as positive (+) and negative (-) control, respectively. (C) Flow cytometric analysis of annexin V was performed by exposing HUVEC and EA.hy926 endothelial lines to doses of LND ranging from 1 to 50 g/ml for 48 hours. The percentage of annexin V- positive cells is reported. Each value is the mean of sextuplicate ±SD, and the data are representative of at least three separate experiments (A and B). A representative experiment out of three is reported (C). Statistical differences between LND-treated versus untreated groups: *P < .05; **P < .01.

Figure 2

LND inhibits HUVEC (black column) and HMVEC (white column) migration (A and C) and invasion (B). Cultured cells were dissociated into a single cell suspension, suspended in DMEM supplemented with 0.1% BSA, and incubated for 6 hours on top of gelatin-coated filters (A and C), or on filters coated with matrigel (B) in the absence (positive and negative control) or presence of LND (1, 5, and 50 g/ml). The migration was evaluated by filling the lower compartment with NIH 3T3 CM (A) or VEGF (C). The invasion was evaluated in response to NIH 3T3 CM (B). DMEM supplemented with 0.1% BSA was used as negative control to evaluate random migration and invasion. The migrated or invaded cells were counted in at least four HPFs (x200). Each value is the mean of triplicates ±SD, and the data are representative of at least three separate experiments. Statistical differences between LND-treated versus untreated groups (positive control): *P < .05; **P < .01.

Figure 3

LND inhibits HMVEC, HUVEC, EA.hy926, and BAEC morphogenesis. Endothelial cells were plated on matrigel in the absence (panel a) or in the presence (panel b) of LND at a dose of 5 g/ml and photographed. Experiments were repeated at least three times, and each dose was tested in triplicate. Representative phase-contrast micrographs are presented.

Figure 4

LND reduces metalloproteases secretion of HUVEC and HMVEC. CM of endothelial cells incubated for 48 hours in SFM in the absence or presence of LND at doses of 5 and 50 g/ml were collected and analyzed for gelatin zymography (A) and Western blotting analysis (B). Proteins from an equivalent number of viable cells were loaded. Red Ponceau staining of filter was used to check equal loadings of proteins. (A) White bands against a dark background correspond to the gelatinolytic areas of MMP2 (72 kDa) and MMP9 (92 kDa) activity. (B) Western blot analysis of MMP2 and MMP9 proteins in HUVECs. Representatives of two independent experiments are shown.

Figure 5

LND reduces vessel formation in a matrigel plug. Vessel formation was assessed after injection of C57BL/6 mice with matrigel plugs containing VEGF alone (positive control) or in combination with two different doses of LND (1 and 5 g/ml). After 5 days, mice were sacrificed, and neovascularization and Hb content of matrigel pellets were evaluated. (A) Macroscopic analysis of matrigels from one representative experiment. (B) Hb content of matrigel plugs. The negative control contained heparin alone. The values were expressed as OD/100 mg of matrigel plug. The OD for each matrigel plug is reported (□). (C) Histologic analysis of matrigel plugs from control mice (panel a) reveals a marked capillary vessel proliferation, as compared with animals treated with LND (panel b). (Masson Trichrome stain; original magnification, x10).

Figure 6

Low doses of LND do not inhibit tumor cell proliferation (A) and do not induce apoptosis (B). ADFS, CG5, JR8, H460, and cells were exposed for 48 hours to doses of LND ranging from 1 to 50 g/ml. (A) Cell proliferation of JR8 (black column), CG5 (dark gray column), ADFS (pale gray column), and H460 (white column) cells was evaluated after incubation of cells in 10% FCS in the absence (control) or in the presence of LND. Each value is the mean of sextuplicates ±SD, and the data are representative of at least three separate experiments. Statistical differences between LND-treated versus untreated groups (positive control): *P < .05; **P < .01. (B) Flow cytometric analysis of annexin V. The percentage of annexin V- positive cells is reported. A representative experiment out of three is reported.

Figure 7

LND does not inhibit metalloproteases secretion, migration, and invasion of tumor cells. CM of tumor cells incubated for 48 hours in SFM in the absence or presence of LND at doses of 5 and 50 g/ml were collected and analyzed for MMP9 and MMP2 gelatin zymography (A) at an equivalent number of viable cells. Representative experiments out of two are reported. Migration (B) and invasion (C) of untreated or LND-treated JR8 melanoma cells in response to NIH 3T3 CM. The migrated or invaded cells were counted in at least four HPFs (x200). Each value is the mean of quadruplicates ±SD, and the data are representative of at least three separate experiments.