A Marine λ-Oligocarrageenan Inhibits Migratory and Invasive Ability of MDA-MB-231 Human Breast Cancer Cells through Actions on Heparanase Metabolism and MMP-14/MMP-2 Axis

Abstract

Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE "modulator" capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels.

Keywords: MDA-MB-231; MMP-14; breast cancer; heparanase; heparin; metalloproteinase; oligosaccharide; polysaccharide; shRNA; λ-carrageenan.

Figure 1

Effects of the four sugars, Unfractionated Heparin (UFH), Low Molecular Weight Heparin (LMWH), native λ-Carrageenan (λ-CAR), and depolymerized λ-CAR (λ-CO) on parental MDA-MB-231 cell viability and proliferation rate. (A) MTT assay was used to analyse cell viability of cells treated with vehicle (control) or compounds at 100 µg/mL for 24 h. (B) Cells were counted after 24 h treatment with either vehicle (control) or compounds at 100 µg/mL. Values are the mean ± SEM of three independent experiments. ANOVA followed by post hoc Fisher’s LSD test. ns: non-significant. Control: parental MDA-MB-231 cell line.

Figure 2

Inhibitory effects of the four sugars on MDA cell migration and invasion. (A) Representative microphotographs and (B) Quantification of MDA cell migration upon vehicle (control), compounds (100 µg/mL) or OGT 2115 (5 µM) treatment for 24 h. Scale bar in A: 100 μm. (C) Representative microphotographs and (D) quantification of MDA cell invasion upon vehicle (control) or compounds (100 µg/mL) treatment for 24 h. Scale bar in C: 100 μm. Values are the mean ± SEM of four (A,B) or three (D) independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; ANOVA followed by post hoc Fisher’s LSD (for B,D). # p < 0.05; ## p < 0.01; ns: non-significant for (B,D); ANOVA followed by post hoc Fisher’s LSD between λ-CAR and λ-CO.

Figure 3

Effects of the four sugars on sh-MDA cells. (A) Analysis by RT-qPCR of HPSE gene expression 48 h after transfection of MDA-MB-231 (parental cells); MDA: transfected with a scramble shRNA; sh-MDA: transfected with sh-RNA against HPSE. (B) WB analysis of HPSE levels on MDA and sh-MDA cells and the corresponding quantifications normalised with β-actin. wcl: whole cell lysates. (C) Effects of the compounds on proliferation rate. sh-MDA cells were counted after treatment with either vehicle (control) or compounds at 100 µg/mL for 24 h. (D) MTT assay was used to analyse cell viability of sh-MDA cells treated with vehicle (control) or treated with compounds at 100 µg/mL for 24 h. (E) WB analysis of LC3 levels in MDA compared with sh-MDA and the corresponding quantifications normalised with β-actin. Ponceau S staining is shown as loading control. Values are the mean ± SEM of three independent experiments. *: p < 0.05; **: p < 0.01; ***: p < 0.005; ns: non-significant. ANOVA followed by post hoc Fisher’s LSD for (A,C,E); Student t test for (B). IB: immunoblot.

Figure 4

Inhibitory effects of the four sugars on sh-MDA cell migration and invasion. (A) Representative microphotographs and (B) Quantification of sh-MDA cell migration upon vehicle (control), compounds (100 μg/mL) or OGT 2115 (5 µM) treatment for 24 h. Scale bar in A: 100 μm. (C) Representative microphotographs and (D) quantification of sh-MDA cell invasion upon vehicle (control) or compounds (100 μg/mL) treatment for 24 h. Scale bar in (D): 100 μm. Values are the mean ± SEM of four (A,B) or three (D) independent experiments. * p < 0.05; ** p < 0.01; ns: non-significant; for (B,D); ANOVA followed by post hoc Fisher’s LSD (for B,D).

Figure 5

(A) Analysis by Western blot of HPSE levels in MDA and MCF-7 cells and the corresponding quantifications normalised with β-actin. Ponceau S staining is showed as loading control. (B) Quantification of MCF-7 migration upon vehicle (control), compounds (100 µg/mL), or OGT 2115 (5 µM) treatment for 24 h. Data are shown as the mean ± SEM of three independent experiments. Values are the mean ± SEM of three independent experiments. ns: non-significant, for (B), ANOVA followed by post hoc Fisher’s LSD. * p < 0.05; ns: non-significant Student t test for (A). IB: immunoblot.

Figure 6

Analyses of the expression of HPSE (A) and cathepsin L (B) by RT-qPCR after 24 h treatment of MDA cells with UFH and λ-CO at 100 µg/mL. (C) Western blot analyses of HPSE after 24 h treatment of UFH and λ-CO at 100 µg/mL and the corresponding quantifications normalised with β-actin of active HPSE (D) and pro-HPSE (E) in whole cell lysates (wcl) and cell supernatants (sup) after 24 h treatment of UFH and λ-CO at 100 µg/mL. The red dotted line indicates splicing of non-adjacent lanes in the same WB. Ponceau S staining is showed as the loading control. (F) Ratio of pro-HPSE sup/HPSE wcl. Values are the mean ± SEM of three independent experiments. * p < 0.05, *** p < 0.005; ns: non-significant, ANOVA followed by post hoc Fisher’s LSD. Control: MDA cells untreated. IB: immunoblot.

Figure 7

Analyses of the expression of MMP2 (A) and MMP9 (B) by RT-qPCR after 24 h treatment of MDA cells by UFH and λ-CO at 100 µg/mL. (C) Gelatin gel zymography images. Analyses of the activity of pro-MMP-9 (D), pro-MMP-2 (E), and MMP-2 (F) after 24 h treatment of UFH and λ-CO at 100 µg/mL. The white dotted line in C indicates splicing of non-adjacent lanes in the same zymogel. Data are shown as the mean ± SEM of three independent experiments. Values are the mean ± SEM of three independent experiments. * p < 0.05; **** p < 0.001; ns: non-significant, ANOVA followed by post hoc Fisher’s LSD. Ctl: Control: MDA cells untreated. ns: non-significant.

Figure 8

Analysis of the expression of TIMP2 (A) and MMP14 (B) encoding genes by RT-qPCR after treatment of MDA cells by UFH and λ-CO at 100 µg/mL for 24 h. (C) Analysis of MMP-14 levels in whole cell lysates after treatment of UFH and λ-CO at 100 µg/mL and the corresponding quantifications normalised with β-actin. The red dotted line indicates splicing of non-adjacent lanes in the same WB. Ponceau S staining is showed as the loading control. Data are shown as the mean ± SEM of three independent experiments. Values are the mean ± SEM of three independent experiments. * p < 0.05; ns: non-significant ANOVA followed by post hoc Fisher’s LSD. Ctl: Control: MDA cells untreated. IB: immunoblot.

Figure 9

(A) Analysis of the expression of MMP-14 encoding gene by RT-qPCR on sh-MDA cells. (B) Gelatin gel zymography images and (C) Quantification of MMP-2 activity on MDA cells (control) compared to sh-MDA cells. (D) Gelatin gel zymography images showing level of MMP-2 in MDA cells compared to MCF-7 cells. (E) Quantification of MMP-2 activity on MDA cells (control) compared to MCF-7 cells. (F) Quantification of pro-MMP-9 levels in MDA cells (control) compared to sh-MDA and MCF-7 cells. Data are shown as the mean ± SEM of three independent experiments. Values are the mean ± SEM of three independent experiments. *: p < 0.05; **: p < 0.01. Student t test for (A,C). Control: MDA cells untreated. ns: non-significant. ANOVA followed by post hoc Fisher’s LSD for (F).