Figure 4
From: Molecular target based combinational therapeutic approaches in thyroid cancer
Estrogen enhanced HUVEC migration, proliferation and tubulogenesis are attenuated by DIM. (A) Conditioned medium was generated by culturing thyroid cancer cells with 10-8 M E2 ± 25 μM DIM or left untreated for 24 hours. HUVECs were allowed to migrate towards thyroid cancer cell conditioned medium used as chemoattractant. The HUVECs that migrated and adhered on the lower surface of the membrane were fixed, stained, and counted in 10X field. The groups are as follows- HUVECs migrated to the untreated (white bars), E2 treated (grey bars), E2 and fulvestrant treated (dotted bars), 25 μM DIM (black bars) and 25 μM DIM + E2 conditioned medium (striped bars). Data expressed as numbers of HUVECs counted (migrated cells) per 10X field micrograph for each sample well and normalized to the untreated control. (B) HUVECs were cultured in presence of thyroid cancer cell conditioned medium followed by trypan blue exclusion cell count to calculate endothelial cell proliferation. The groups are as follows- HUVECs migrated to the untreated (white bars), E2 treated (grey bars), E2 and fulvestrant treated (dotted bars), 25 μM DIM (black bars) and 25 μM DIM + E2 conditioned medium (striped bars). Data expressed as % of HUVECs cell number normalized to the untreated control. The asterisk denotes statistically significant differences (p < 0.05) between experimental and untreated group using one way ANOVA tests. (C) Conditioned medium was generated by culturing ML-1 cells with estrogen ± DIM for 24 hours. Growth factor-reduced Matrigel was plated and allowed to polymerize in 96-well plates. HUVECs were seeded in ML-1 thyroid cancer cell conditioned medium on matrigel. The plate was incubated for four to six hours and the wells were photographed using an inverted microscope with a digital camera. The results are representative of three independent experiments performed in triplicates.