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. 2018 Apr 25:24:13.
doi: 10.1186/s40409-018-0150-2. eCollection 2018.

Alternagin-C binding to α2β1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Milene Nóbrega de Oliveira Moritz  1 , Lívia Mara Santos Eustáquio  1 , Kelli Cristina Micocci  1 , Ana Carolina Caetano Nunes  1 , Patty Karina Dos Santos  1 , Tamires de Castro Vieira  1 , Heloísa Sobreiro Selistre-de-Araujo  1
Affiliations

Alternagin-C binding to α2β1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Milene Nóbrega de Oliveira Moritz et al. J Venom Anim Toxins Incl Trop Dis. .

Abstract

Background: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2β1 integrin. Herein, we used ALT-C as a α2β1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration.

Methods: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2β1 integrin binding properties of ALT-C, its dissociation constant (Kd ) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting.

Results: Our data demonstrate that ALT-C, after binding to α2β1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression.

Conclusion: These results demonstrate that α2β1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.

Keywords: ALT-C; C-Myc; Cancer; MMP; Tumor microenvironment; α2β1 integrin.

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Conflict of interest statement

Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characteristics of ALT-C binding to α2β1 integrin or collagen type I (Col I). Representative sensorgrams and dose-dependent binding of ALT-C (0.0–5.0 μM) measured by surface plasmon resonance (SPR): (a) to α2β1 integrin; and (b) to Col I. Kinetic curves were analyzed using the 1:1 binding model by GraFit 7 software for: (c) α2β1 integrin; and (d) Col I
Fig. 2
Fig. 2
ALT-C stimulates the transmigration of MDA-MB-231 cells through a monolayer of HMEC-1 cells. a MDA-MB-231 cells were plated in wells containing HMEC-1 cells, and 5% of fetal bovine serum (FBS) was used as a chemoattractant in the lower chamber. Tumor cells were first stained with PKH26 Red Fluorescent Cell Linker and after 16 h of transmigration assay the cells were fixed, stained with DAPI and counted (an average of eight fields from each treatment). Negative control means the assay in the absence of FBS in the lower chamber. The assay was performed in triplicate with two independent assays (n = 2). The results were compared using ANOVA followed by Tukey’s test (* p < 0.05, and *** p < 0.001). b Representative images of transmigrated cells of each treatment. Bar represents 50 μm
Fig. 3
Fig. 3
ALT-C effects on: (a) MDA-MB-231 cells; (b) fibroblasts; and (c) HMEC-1 cells were plotted as a percentage of wound closure 24 h after wounding. FBS 10% represents cells in presence of medium with FBS (10%) as positive control. The assay was performed in triplicate with two independent assays (n = 2). p values were determined using ANOVA followed by Tukey’s test, considering significant when p < 0.05. Representative photos of wounds were taken at time zero and 24 h after wounding. Cells were stained with crystal violet 0.1%
Fig. 4
Fig. 4
Effects of ALT-C on the MMP-9 and MMP-2 contents in the conditioned media from (a) MDA-MB-231, (b) human fibroblasts, and (c) HMEC-1 cells. MMP content was detected by band densitometry in the conditioned media (peak area) after wounding and incubation with ALT-C for 24 h. MMP-2 and MMP-9 contents were determined by band densitometry. The assay was performed in triplicate with two independent assays (n = 2). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control (without ALT-C)
Fig. 5
Fig. 5
(a) ALT-C effects on MMP-9 expression in MDA-MB-231 cells, and (b) on MMP-2 mRNA levels in fibroblasts or (c) HMEC-1 cells. Levels of c-Myc mRNA after ALT-C treatment in (d) MDA-MB-231 cells, (e) human fibroblasts, and (f) HMEC-1 cells. The values represent relative transcript abundance and the p value was determined using ANOVA followed by Tukey’s test. Values were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The assay was performed in triplicate with two independent assays (n = 2). * p < 0.05, *** p < 0.001 compared to control (0 nM)
Fig. 6
Fig. 6
ALT-C induces FAK phosphorylation at 10 nM but not at 100 and 1000 nM. a FAK and p-FAK protein level revealed by Western blotting in lysates extracted from MDA-MB-231 treated with ALT-C (10, 100 and 1000 nM) and control (0 nM). b Values represent the normalized densitometry ratio of p-FAK and FAK and the p value was determined using ANOVA followed by Tukey’s test. Values were also previously normalized to the level of GAPDH densitometry. ** p < 0.01 compared to control (0 nM). The assay was performed in independent assays (n = 2) and Western blotting in quadruplicate
Fig. 7
Fig. 7
Potential mechanism of ALT-C in the tumor microenvironment and cancer progression. (1) Transmigration of breast tumor cells (in blue) through endothelial cells is induced by low concentrations of ALT-C. (2) ALT-C binds to α2β1 integrin, triggering c-Myc upregulation via p-FAK and p-AKT activation resulting in downregulation of the proteins (3) MMP-9 and (4) MMP-2, thus decreasing tumor cell invasion into ECM. ALT-C also acts via α2β1 integrin on endothelial cells, decreasing MMP-2, which inhibits the formation of new blood vessels
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