Asiatic Acid Inhibits Pro-Angiogenic Effects of VEGF and Human Gliomas in Endothelial Cell Culture Models
Figure 5
AsA inhibits human glioma cell-induced angiogenesis in vitro.
A. LN18 and U87-MG human glioma cells were treated with AsA at 20 µM doses for 24 h, media was removed, washed with 0.5% serum media and incubated for an additional 12 h in 0.5% serum media without the presence of DMSO or AsA. Subsequently, both LN18 and U87-MG cells were trypsinized, and counted using haemocytometer as detailed in ‘Materials and Methods’. B. VEGF expression in CCM (control conditioned media) and AsA20CM (AsA20 conditioned media) or total cell lysates collected from LN18 and U87-MG cells (detailed in ‘Materials and Methods’) was analyzed by Western blotting. The loading volume for conditioned media in each case was normalized with respective cell number. Densitometric values presented below the bands are ‘fold change’ compared to respective controls. AsA20-T/W refers to the group, where glioma cells were treated with AsA 20 µM dose for 24 h and then AsA was washed-out and cell lysates were prepared after 12 h. C & D. HUVEC or HBMEC (4×104 per well) were seeded in 24-well plates coated with matrigel and treated with CCM or AsA20CM from LN18 cells or U87-MG cells for 10 h and tube formation assay was performed as described in ‘Materials and Methods’. In this experiment, HUVEC or HBMEC incubated with 0.5% serum containing LN18 or U87-MG media served as a negative control. Tubular structures were photographed at 100x magnification and tube length was measured as described in ‘Materials and Methods’. Tube length data is presented as mean ± standard deviation of three samples for each treatment. The volume for CCM/AsA20CM used in tube formation assay was normalized with respective cell number shown in panel A. *, p≤0.001.