The Effects of αvβ3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro

Abstract

Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvβ3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvβ3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvβ3 integrin during tumor progression.

Keywords: DisBa-01; breast tumor; cell migration; disintegrin; hypoxia; αvβ3 integrin blocking.

Figure 1

Inhibition of MDA-MB-231 cell migration by αvβ3 integrin blocking in normoxia and hypoxia. (A–C) Boyden chamber assay, MDA-MB-231 cells treated with indicated DisBa-01 concentrations. (A) Migrated cells in absence or presence of DisBa-01 in normoxia and hypoxia. Values were compared to negative control (without chemoattractant). (B) Representative images of migrating cells treated or not with DisBa-01 in hypoxia. (D–F) Transendothelial migration of CFSE-labeled MDA-MB-231 cells in a HUVEC layer. (D) Transmigrated cells in absence and presence of DisBa-01 in normoxia and hypoxia. Values were compared to negative control (without chemoattractant). (A,D) graphics represent mean ± SD. (E) Representative images of transmigrating cells treated or not with DisBa-01 in hypoxia. (C,F) Graphical summary of the two assays. (G–M) Wound healing assay of MDA-MB-231 cells in the presence of DisBa-01 in normoxia and hypoxia for 12 (G–H), 24 (I–J), and 48 (K–L) hours. (M) Graphical summary of the wound healing assay in the presence of DisBa-01. (G,I,K) graphics represent median ± SD. Letters over bars mean: a, significantly different from control; b, significantly different from 10 nM; c, from 100 nM; d, from 250 nM, and e, from 500 nM (mean ± SD). All experiments were performed in triplicate from three independent assays (n = 3, p < 0.05). Scale bar: 100 μm. Red arrows in graphical summary represent the direction of migration.

Figure 2

MMP-2 and MMP-9 levels in the conditioned media (CM) from MDA-MB-231 cell migration assays. (A,E,I) Representative zymographs of CM from transwell, transmigration, and wound healing assays; (B,F,J) Quantification of pro-MMP-9 levels by densitometry; (C,G,K,L) Quantification of pro-MMP-2 and active MMP-2 levels by densitometry; (D,H,M), CM total protein concentration. (N) Graphical summary of the assays. Experiments were performed in triplicate with three independent assays (n = 3). The results (mean ± SD) were compared using two-way ANOVA followed by Tukey’s test (p < 0.05). Graphic letters a, b, c, d, and e represent comparisons among 0, 10,100, 250, 500, and 1000 nM of DisBa-01, respectively.

Figure 3

Inhibition of tube formation and cell migration of DisBa-01-treated HUVECs in normoxia and hypoxia. (A–F) Tube formation assay. Representative images of HUVECs in the indicated conditions (A), total length (B), master junctions (C), number of nodes (D), and score (E) by pixel quantification of DisBa-01-treated HUVECs in the two oxygen conditions. Experiments were performed in triplicate of three independent assays (n = 3, p < 0.05). (F) Graphical summary of the assay. (G–H), Boyden chamber migration assay of DisBa-01-treated HUVECs. Values were compared to negative control (without chemoattractant) (G). Representative images of migrating cells treated or not with DisBa-01 in hypoxia (H). Scale bar: 100 µm. (J–N) HUVEC wound healing assay. Percentage (mean ± SD) of wound opening in indicated concentrations of DisBa-01 in normoxia and hypoxia after 9 and 24 h (J,L). Representative images of scratches in hypoxia (K,M). (I,N) Graphical summary of the two migration assays. Graphic letters a, b, c, d, e, and f represent comparisons between 0, 10,100, 250, 500, and 1000 nM of DisBa-01, respectively. All experiments were performed in triplicate from three independent assays (n = 3, p < 0.05). Red arrows in graphical summary represent the direction of migration.

Figure 4

Profile of β3 integrin subunit in MDA-MB-231 and HUVECs in normoxia and hypoxia. (A–F) Detection of β3 integrin subunit in MDA-MB-231 cells in normoxia (A), in hypoxia (B), and merge of (A) and (B) (C). Detection of β3 integrin in MDA-MB-231 cells after DisBa-01 treatment in normoxia (D), and in hypoxia (E), and negative and positive controls in normoxia and hypoxia (F). (G–L) Detection of β3 integrin subunit in HUVECs in normoxia (G), in hypoxia (H), and merge of (G) and (H); (I) HUVEC β3 integrin content after DisBa-01 treatment in normoxia (J) and in hypoxia (K), and negative and positive controls in normoxia and hypoxia (L). (M) Data quantification and statistics for MDA-MB-231 cells and HUVECs under normoxia and hypoxia. * means statistical differences between MDA-MB-231 cells (green bars) and HUVEC (gray bars) in normoxia and hypoxia. (N) Graphical summary of β3 integrin subunit profile in MDA-MB-231 cells and HUVECs with or without DisBa-01 in normoxia and hypoxia. Experiments were performed in triplicate from three independent assays (n = 3, p < 0.05).

Figure 5

The morphology of MDA-MB-231 cells and HUVEC changes upon αvβ3 integrin blocking by DisBa-01 in normoxia and hypoxia. (A) MDA-MB-231 cells treated with DisBa-01 for 4 h. (B) Graphic represents the sum of cell total area (µm2) divided by the number of nuclei in the two conditions. Analysis was performed using ImageJ after cell staining in 100 cells per well. (D) HUVECs were treated with DisBa-01 for 4 h. (E) Graphic represents cell area (µm2) divided by the number of nuclei in normoxia and hypoxia. Experiments were performed in duplicate or triplicate with three independent assays (n = 3). Results for MDA-MB-231 cells were compared using two-way ANOVA followed by Tukey’s test (p < 0.05). HUVEC results were compared using the Kruskal–Wallis one-way analysis of variance on ranks post hoc Dunn test and all data (p < 0.05). Results are shown as median with range of variation. Graphic letters a, b, c, and d represent comparisons among 0, 100, 1000, and 2000 nM of DisBa-01, respectively. Scale bar = 50 μm. Graphical summary of MDA-MB-231 (C) and HUVEC (F) morphology with and without DisBa-01 in normoxia and hypoxia.