Antiproliferative Activity of Glycosaminoglycan-Like Polysaccharides Derived from Marine Molluscs

Abstract

Despite the increasing availability of new classes of cancer treatment, such as immune- and targeted therapies, there remains a need for the development of new antiproliferative/cytotoxic drugs with improved pharmacological profiles that can also overcome drug resistant forms of cancer. In this study, we have identified, and characterised, a novel marine polysaccharide with the potential to be developed as an anticancer agent. Sulphated polysaccharides isolated from the common cockle (Cerastoderma edule) were shown to have antiproliferative activity on chronic myelogenous leukaemia and relapsed acute lymphoblastic leukaemia cell lines. Disaccharide and monosaccharide analysis of these marine polysaccharides confirmed the presence of glycosaminoglycan-like structures that were enriched in ion-exchange purified fractions containing antiproliferative activity. The antiproliferative activity of these glycosaminoglycan-like marine polysaccharides was shown to be susceptible to heparinase but not chondrotinase ABC digestion. This pattern of enzymatic and antiproliferative activity has not previously been seen, with either marine or mammalian glycosaminoglycans. As such, our findings suggest we have identified a new type of marine derived heparan sulphate/heparin-like polysaccharide with potent anticancer properties.

Keywords: anticancer; antiproliferative; glycosaminoglycans; heparan sulphate; marine mollusc.

Figure 1

Antiproliferative activity of cockle polysaccharides on cancer cell lines. Two cancer cell lines K562 (A) and MOLT4 (B) were treated with increasing doses of cockle polysaccharides and cell viability was determined by MTT assay, as detailed in the “Materials and Methods” section. Inserts show effects of cisplatin treatment on viable cell number. Cells were cultured under standard conditions and maintained at 37 °C in a humidified 5% CO₂ atmosphere. Cell viability is expressed as a percentage relative to untreated control cells. All experiments were conducted in triplicate and the IC₅₀ values were calculated using non-linear regression analysis (GraphPad Prism 5.0).

Figure 2

Cell cycle analysis and Annexin V apoptosis assay. MOLT4 cells were treated with 50 μg of cockle polysaccharides for 24 h then stained using Annexin V conjugated Fluorescein isothiocyanate (Annexin V-FITC) and/or PI. (A) Flow cytometry cell cycle analysis of PI stained cells with or without cockle polysaccharide treatment. A single representative experiment is shown. (B) Flow cytometry scatter plot of Annexin V-FITC/PI stained cells with or without cockle polysaccharide treatment. A single representative experiment is shown. (C) Quantitative cell cycle analysis as determined by PI staining and flow cytometry. The percentage of cells in each phase is shown for control (☐) and cockle polysaccharide treated (■) cells. Results are presented as the mean ± SD of three independent experiments. Statistical significance was determined using the two-tailed Student’s t-test. p < 0.05 was considered statistically significant (*). (D) Quantitative analysis of apoptosis as determined by Annexin V-FITC/PI staining and flow cytometry. The percentage of cells in each quadrant is shown for control (☐) and cockle polysaccharide treated (■) cells. Results are presented as the mean ± SD of three independent experiments. Statistical significance was determined using the two-tailed Student’s t-test. p < 0.05 was considered statistically significant (*).

Figure 3

Effect of heparinase (I, II, and III) and chondroitinase ABC enzymatic degradation on cockle polysaccharide antiproliferative activity. Sensitivity of the cockle polysaccharide antiproliferative activity to enzymatic degradation was determined by MTT assay. Antiproliferative activity of cockle polysaccharides on K562 cells (A) and MOLT4 cells (B), with and without heparinase or chondrotinase ABC digestion. Intact cockle polysaccharides (◯), heparinase I treated (∆), heparinase II treated (▲), heparinase III treated (■), heparinase I, II, and III treated (□) and chondroitinase ABC (●). The data are presented as the percentage of viable cells following treatment with cockle polysaccharides, relative to untreated control. All experiments were conducted in triplicate and the results are shown as the mean ± the SD. Cells were cultured in suspension and maintained at 37 °C in humidified 5% CO₂ atmosphere.

Figure 4

Comparison of mammalian GAGs and cockle polysaccharides antiproliferative activity. Differences in biological activities of mammalian GAGs and cockle polysaccharides on K562 (A) and MOLT4 (B) cell lines was assessed by MTT assay, cockle polysaccharides (◯), mammalian HS (☐). Data are presented as the percentage of viable cells following treatment with cockle polysaccharides, relative to untreated control. All experiments were conducted in triplicate and the results are shown as the mean ± the SD. Cells were cultured in suspension and maintained at 37 °C in humidified 5% CO₂ atmosphere.

Figure 5

Ion-exchange chromatography of cockle polysaccharides. Cockle polysaccharides were applied to a DEAE-Sepharose column and eluted using a 0–1.5 M NaCl gradient over 70 min. Peaks were pooled as indicated by the bars shown, lyophilised and Fractions 1–6 stored at −20 °C for further analysis.

Figure 6

Antiproliferative properties of the ion-exchange purified cockle polysaccharide fractions. Measurement of antiproliferative activity of ion-exchanged purified fractions on K562 (A) and MOLT4 (B) cells was achieved by MTT assay. Fraction 1 (◯), Fraction 2 (☐), Fraction 3 (∆), Fraction 4, (■), Fraction 5 (▲) and Fraction 6 (●). The data are presented as the percentage of viable cells following treatment with cockle polysaccharides, relative to untreated control. All experiments were conducted in triplicate and the results are shown as the mean ± the SD. Cells were cultured in suspension and maintained at 37 °C in humidified 5% CO₂ atmosphere.