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Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H 2 O 2 -assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (M n and M w ) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H 2 O 2 -based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

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.

In tumor development, the degradation of heparan sulfate (HS) by heparanase (HPSE) is associated with cell-surface and extracellular matrix remodeling as well as the release of HS-bound signaling molecules, allowing cancer cell migration, invasion and angiogenesis. Because of their structural similarity with HS, sulfated polysaccharides are considered a promising source of molecules to control these activities. In this study, we used a depolymerisation method for producing λ-carrageenan oligosaccharides (λ-CO), with progressive desulfation over time. These were then used to investigate the influence of polymeric chain length and degree of sulfation (DS) on their anti-HPSE activity. The effects of these two features on λ-CO anticoagulant properties were also investigated to eliminate a potential limitation on the use of a candidate λ-CO as a chemotherapeutic agent. HPSE inhibition was mainly related to the DS of λ-CO, however this correlation was not complete. On the other hand, both chain length and DS modulated λ-CO activity for factor Xa and thrombin IIa inhibition, two enzymes that are involved in the coagulation cascade, and different mechanisms of inhibition were observed. A λ-carrageenan oligosaccharide of 5.9 KDa was identified as a suitable anticancer candidate because it displayed one of the lowest anticoagulant properties among the λ-CO produced, while showing a remarkable inhibitory effect on MDA-MB-231 breast cancer cell migration.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC 50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a Matrigel TM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 μg·mL⁻¹ (0.17 μM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 μg·mL⁻¹ (13.3 μM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 40 μg·mL⁻¹ (66.7 μM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained.