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. 2019 Jul 10;19(12):978–985. doi: 10.1002/elsc.201900019

Characterization and potential antitumor effect of a heteropolysaccharide produced by the red alga Porphyridium sordidum

Biliana Nikolova 1, Severina Semkova 1, Iana Tsoneva 1, Georgi Antov 1, Juliana Ivanova 2, Ivanina Vasileva 2, Proletina Kardaleva 3, Ivanka Stoineva 3, Nelly Christova 4, Lilyana Nacheva 4, Lyudmila Kabaivanova 4,✉
PMCID: PMC6999067  PMID: 32624987

Abstract

Taking into account the rising trend of the incidence of cancers of various organs, effective therapies are urgently needed to control human malignancies. However, almost all chemotherapy drugs currently on the market cause serious side effects. Fortunately, several studies have shown that some non‐toxic biological macromolecules, including algal polysaccharides, possess anti‐cancer activities or can increase the efficacy of conventional chemotherapy drugs. Polysaccharides are characteristic secondary metabolites of many algae. The efficacy of polysaccharides on the normal and cancer cells is not well investigated, but our investigations proved a cell specific effect of a newly isolated extracellular polysaccharide from the red microalga Porphyridium sordidum. The investigated substance was composed of xylose:glucose and galactose:manose:rhamnose in a molar ratio of 1:0.52:0.44:0.31. Reversible electroporation has been exploited to increase the transport through the plasma membrane into the tested breast cancer tumor cells MCF‐7 and MDA‐MB231. Application of 75 µg/mL polysaccharide in combination with 200 V/cm electroporation induced 40% decrease in viability of MDA‐MB231 cells and changes in cell morphology while control cells (MCF10A) remained with normal morphology and kept vitality.

Keywords: algal heteropolysaccharide, antitumor effect, Porphyridium sordidum

Abbreviation

DW

dry weight

1. INTRODUCTION

Cancer is one of the most common reasons for human mortality. The treatment of cancer diseases requires a therapeutic approach with severe side effects. Recently, new sources of non‐toxic natural substances with potential anticancer effects are under active investigation 1. Marine resources attract the attention of many researchers focusing on the diverse biological activities of microalgal polysaccharides and the possibilities for their application 2. There has been a tremendous interest in developing anti‐cancer polysaccharide drugs over the last decade. Polysaccharides from renewable sources as fungi or algae belong to biomacromolecules in which residues of monosaccharides are connected to each other by glycosidic linkages. They offer some biological information due to the structural variability comparing with nucleic acids or proteins 3. Polysaccharides are characteristic secondary metabolites of many algae. Although cultivation of microalgae seems easy, there are many challenges including minimizing contamination, efficient provision of carbon dioxide, controlled light supply following optimal cultivation conditions, in that way reducing production costs, as well as minimizing space requirements. Microalgae can produce large amounts of extracellular polysaccharides, thanks to the improvements in the algal biotechnology obtaining a high yield under controlled cultivation conditions 4. Another advantage is the possibility for easy and efficient isolation of these exopolysaccharides from the algal cultures 5. Main producers of these compounds are red microalgae 6. These marine natural products have other advantages due to their availability, low toxicity, as well as having a great variety of mechanisms of action 7. Algal polysaccharides can possess either a direct inhibitory action on cancer cells and tumors or influence different stages of carcinogenesis and tumor development, recover the broken balance between proliferation and programmed cell death (apoptosis), and are useful for cancer prophylactics. The essence of the anti‐cancer mechanism is in the ability of the polysaccharides to inhibit tumor angiogenesis as well as to stimulate the immune response and induce apoptosis 8. Polysaccharides possess a wide range of biological activities on inflammation, cell–cell recognition, transduction and immune responses, and anti‐cancer activity. The protective effect of Dixoniella grisea (Rhodophyta) polysaccharide on Graffi myeloid tumor in hamsters showed the various doses and ways of application of polysaccharide administered inhibition of tumor growth 9. The extracellular polysaccharides of Porphyridium cruentum consisted mainly of xylose, galactose, and glucose. They inhibited the growth of the implanted S180 tumor via immunoenhancement 10. The efficacy of polysaccharides on the normal and cancer cells is not well investigated and could be cell specific due to the differences in their surface structure and metabolism. On the other hand, the reversible electroporation has been exploited to increase transport of chemotherapeutic drugs or nano particles (electrochemotherapy) through the plasma membrane into the tumour cells 11, 12. The application of electroporation in the presence of big polysaccharides molecules could have some additional or synergetic effect.

PRACTICAL APPLICATION

Cultivating and harvesting products from microalgae has led to increasing interest in their use for producing valuable substances for food, feed, cosmetics, pharmacy, and medicine. Microalgae are unique and potentially valuable microorganisms because they are light‐harvesting and can convert carbon dioxide into biomass or a variety of bioactive compounds. Finding newly isolated compounds from microalgae with a uniquely high effective action in the treatment of tumors and with no toxic effect on normal cells would be a prerequisite for deepening the research and including in vivo experiments for the treatment of some forms of cancer, and ultimately improving health care for people.

The purpose of our study was to isolate and characterize an extracellular heteropolysaccharide from the red microalgal strain Porphyridium sordidum and test its potential properties as a natural, safe bioactive product to be used as an alternative for treatment of breast cancer cells stepping on algological, biophysical, and biomedical approaches.

2. MATERIALS AND METHODS

2.1. Algae cultivation

Monoalgal, non‐axenic cultures of Porphyridium sordidum (Geitler.) OTT 1966/SAG 114.79 (Rhodophyta) from the culture collection of the Institute of Botany (ASCR, Třeboň, Czech Republic) were used. Intensive cultivation of microalgae was performed in 200 mL special glass vessels at 26°C under continuous illumination (white fluorescent light, 132 µmol/m2/s). A carbon source was provided by bubbling sterile 2% v/v CO2 in air through the cultures. The strains were cultured in appropriate liquid medium 13. The initial density of the cultures was 0.8 mg/mL.

Specific growth rate (µ, d−1) was calculated from the dry weight (DW): µ (N 2/N 1)/t 2‐t 1, where N 1 and N 2 are the DWs of algal cells at the definite time periods‐ t1 and t2.

2.2. Isolation and purification of algal polysaccharide

The isolation from Porphyridium sordidum was carried out according to Simon et al. 1992 14. Cultures at the stationary phase of growth were centrifuged (18 000 x g) and the supernatant containing the soluble extracellular polysaccharide was mixed with 96% ethanol (1:1, v/v). The gel‐like precipitate was collected, dissolved, and dialyzed (2.3 cm dialysis tubing, MW cutoff 12.4 kDa) against distilled water. Dialysis took place for 48 h at 4°C. The obtained polysaccharide solution was sterilized through a bacterial filter (0.2 µm), then dried by lyophilization and powdered for testing in the present experiments.

Measurement of viscosity (ɳ) of extracellular polysaccharide from Porphyridium sordidum was accomplished for quantity estimation. The viscosity (ɳ) of the polysaccharide was measured by viscometer V3 (DIN 53015). It was calculated after the time of passage of the measuring sphere between two marks. It was a mean value from five repetitions.

Algal growth was followed. For DW determination, biomass was centrifuged, washed, and dried at 105°C and the algal density was estimated gravimetrically.

2.3. Identification of crude polysaccharide

The crude biomass after partial or total acid hydrolysis was analysed by TLC. The sample was dissolved in methanol and spotted on silica gel TLC plate (TLC Silica gel 60 F254, Merck Darmstadt, Germany). The plate was developed with a mobile phase of ethanol/1‐butanol/water/acetic acid respectively in ratio 10:1:3:3 v/v/v/v and detected with a 0.2% solution of orcinol in aqueous 20% H2SO4, at 100‐l10°C 15. The monosaccharide composition including Galacturonic acid and Glucoronic acid was performed with 2 M TFA for 24 h at 80°C by sonification.

2.4. Assays of antitumor properties

2.4.1. Cell lines and polysaccharide treatment

Two human breast cancer cell lines (MCF‐7 low metastatic; MDA‐MB231 high metastatic) were cultivated in RPMI‐1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum and 2 mM L‐glutamine. Non‐cancer human breast cell line MCF10A was cultivated in DMEM/F12 (Invitrogen 11330‐032), supplemented with horse serum (Invitrogen#16050‐122), essential growth factor, hydrocortisone cholera Toxin, Insulin. All cell lines were maintained at 37°C in an incubator with humidified atmosphere containing 5% CO2 and were routinely passaged when 80–85% of cells were confluent using 0.25% trypsin and 0.02% EDTA (Invitrogen, Karlsruhe, Germany).

2.4.2. Electroporation

An electroporator Chemopulse IV, generating bipolar pulses, was used in the experiments. The instrument is equipped with a large voltage control in the limits of 100–2200 V, simplified operations, locking against illegal manipulations, and enhanced protection against electrical hazards. The electrotreatment was done by 16 biphasic pulses, each of them 50 + 50 µs duration with 20 µs pause between both phases and pause between bipolar pulses of 880 µs. In each experiment, parallel stainless steel electrodes were used. The intra‐electrode distance was 1 cm. Electric pulses with intensity of 200 V/cm were applied.

Electroporation protocol: cells (1 × 105 cells per well) were seeded 24 h before electroporation. Polysaccharides in concentrations 10, 25, 50, 75, and 100 µg/mL were added immediately before pulse application. The controls were treated under the same conditions, but without electric pulse application and/or addition of polysaccharides.

2.4.3. Cells viability assay

The viability of cells was determined by MTS [Owen's reagent: 3‐(4, 5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxy phenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt]. The MTS‐test was applied after cells treatment with electrical pulses in the absence or presence of polysaccharides.

2.4.4. Observation of cell morphology changes

To evaluate the effect of polysaccharides treatment on the morphology of cells, 1 × 105 MDA‐MB231 cells were seeded in six well plates and expose to the 75 µg/mL polysaccharide (cell line and concentration with highest effect) for 48 h. The morphology changes were documented with light microscope Carl Zeiss, Jena.

2.4.5. DNA isolation and fragmentation analysis

To investigate the effect of polysaccharide treatment on DNA fragmentation, 1 × 105 MDA‐MB231 cells were seeded in six well plates and exposed to the 75 µg/mL polysaccharide (cell line and concentration with highest effect) for 48 h. Cells were collected and DNA was isolated with Quick‐gDNA™ miniPrep kit (Zymo Research).

2.4.6. Statistical analyses

The experiments were performed in triplicate and presented with the ±SD.

To evaluate the statistical significance of the cell viability reduction, a comparison between exposed and control probes was performed by Student's t‐test. p‐values lower than 0.05 were considered statistically significant. IC 50 was calculated according to Quest Graph™ IC50 Calculator 16.

3. RESULTS AND DISCUSSION

Our previous findings showed that treatment of cancer cells with the heteropolysaccharide from another red algal strain Porphyridium cruentum alone or in combination with electroporation induced reduction in cell viability 17. Stepping on this fact, the research continued. After cultivation, an extracellular polysaccharide was isolated, purified, characterized, and tested for antitumor properties.

3.1. Algal growth and polysaccharide accumulation

A bubble column glass vessel (volume 200 mL) was used for intensification of algal growth and polysaccharide production. It is equivalent to a photobioreactor with controlled supply of CO2, temperature, and light control. Estimation of algal growth showed that after 120 h of intensive cultivation of Porphyridium sordidum, the DW increased six‐fold. At the same time, the viscosity (ɳ) of the culture medium (directly correlating to the quantity of extracellular polysaccharide) reached up to 4.2 mPa/s (three times increase). As seen from Figure 1, polysaccharide accumulation was growth associated.

Figure 1.

Figure 1

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Growth of Porphyridium sordidum and polysaccharide accumulation

Polysaccharide yield and specific growth rate were determined and are presented in Figure 2. Maximal yield was reached at the 120th hour (0.92 g/L). No further increase was observed in the next hours. The highest specific growth rate was registered at the 48th hour. For another red alga Porphyridium purpureum, Li et al. (2019) established the value of 0.299 g/L for exopolysaccharide yield after 16 days of cultivation 18.

Figure 2.

Figure 2

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Polysaccharide yield and specific growth rate of the algal culture

3.2. Chemical characterization

Based on to the conveyed analyses (TLC and HPLC), the composition of the extracellular polysaccharide produced by the red alga Porphyridium sordidum after total hydrolysis revealed mainly xylose, glucose, galactose, rhamnose, manose, and glucuronic acid (Figure 3). Sugar composition analysis by HPLC showed that the investigated sample was composed of Xyl:Glc and Gal:Man:Rha in a molar ratio of 1:0. 52:0.44:0.31.

Figure 3.

Figure 3

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HPLC chromatograms of sugars in hydrolysates sample

Presence of a certain amount of sulphates was also registered (Figure 4). Among the polysaccharides, sulfated polysaccharides are the bioactive macromolecules in which some hydroxyl groups are substituted with sulfate groups in the sugar residues, which are responsible in some cases for their antitumor properties. Vishchuk et al. (2011) isolated fucoidans from brown seaweeds Saccharina japonica and Undaria pinnatifida and tested their antitumor activity against human breast cancer T‐47D and melanoma SK‐MEL‐28 cell lines. They distinctly inhibited proliferation and colony formation in both breast cancer and melanoma cell lines in a dose‐dependent manner 19.

Figure 4.

Figure 4

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Chemical composition of the crude red algae isolated from Porphyridium sordidum

Some authors report a mechanism of anticancer activity by blocking the G0/G1 phase of cancer cells. This result might be related to the differences in the chemical components and structure of the polysaccharides 20.

The standard methods for protein assay according to Lowry 21 and Bradford 22 give negative results. Protein content of the studied biomass was determined through nitrogen measurement by the Dumas method and estimated using nitrogen to protein conversion factor of 6.25, assuming that no non‐protein nitrogen sources were present in sample. The analysis was performed in triplicate. Ash content was determined by standard test method for ash content ASTM D5630‐13 (Figure 4). These results showed that in the crude biomass of Porphyridium sordidum, low amounts of ash and proteins were present.

3.3. Potential antitumor activity

In this study, we examined the anti‐proliferative effect of the isolated polysaccharide. Two different types of cancer cell lines (MCF‐7 low metastatic; MDA‐MB231 high metastatic) and one normal cell line (MCF10A) were tested. The cells were treated with 10, 25, 50, 75, or 100 µg/mL of polysaccharide for 24 and 48 h. The cell proliferation was tested by MTS test assay. The results showed that the polysaccharide cannot affect significantly cell viability 24 h after incubation (data not shown), but 48 h after the cell survival appeared to be dose and cell type dependent (Figure 5A,B). Additional application of electroporation (200 V/cm) increased the effect of polysaccharide on cell proliferation. The IC50 was calculated as 69.062, 32.05, and 171.117 for MCF‐7, MDA‐MB231, and MCF10A, respectively. It is interesting to remark that the effect of treatment is different from one cell line to another. The lowest effect was measured on MCF10A cell line (non‐cancer cell line) (Figure 5C).

Figure 5.

Figure 5

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Cell viability of (A) MCF‐7 human cancer cells; (B) MDA‐MB 231 human cancer cells, and (C) MCF10A non‐cancer human cells after combined treatment with polysaccharide and/or 200 V/cm electrical pulses. Incubation time: 48 h. Note: The data are average from three independent experiments. Bars: SD. *statistically significant (p = <0.05)

As a diverse class of macromolecules, PS play an important role in many biological processes. Recently, some authors demonstrated that polysaccharides had a broad spectrum of biological effects including anticancer activities 23, 24, 25.

Polysaccharides already proved to have several important properties, especially the sulfated ones, including their antitumor, anti‐inflammation, anticancer, and immunomodulation activities 26, 27. However, the attempts to establish a relationship between the structures of PS and their bioactivities/actions had been a challenge due to the complexity of this type of polymers. Recently, some possible mechanisms of anticancer activity of PS were reported by inducing cell apoptosis following a pathway dependent of Caspases‐3 and ‐7 28 by inactivating of the epidermal growth factor (tyrosine kinase) receptor (EGFR), which was greatly involved in cell transformation, differentiation, and proliferation by activating of the expression of Fas/FasL 29, 30.

Here in our study, we treated three different human breast cancer cell lines: MCF‐7 low metastatic, MDA‐MB231 high metastatic, and MCF10A “normal‐like” epithelia cell line. The MCF10A cell line used represented healthy cells to determine the level of safety at possible use of the tested compounds in human subjects.

Some differences between the tested cell lines MCF‐7 and MDA‐MB231 exist. Both are reliable and are very common in breast cancer research. MCF10A “served” as a control of the treatment. MCF‐7 is luminal breast cancer cell line, which means the cells express estrogen receptor, progesterone receptor but not the Her2/Neu receptor. This cell line is often responsive to chemotherapy. These cells still retain some of their differentiated epithelial characteristics such as the ability to form domes and to process estradiol via the estrogen receptor.

MDA‐MB‐231 is a triple negative human breast cancer cell line, lacking all three receptors. These cells are highly aggressive, and do not often respond to chemotherapy. This cell line harbors TP53, BRAF, CDN2A, KRAS, and NF2 gene mutations. They are mesenchyme in shape.

We found dose and time‐dependent manner mechanism with a significant effect on high metastatic cell line and almost no effect on normal cell line. The application of electrotreatment further enhanced the therapy effect, as evidenced by a reduction in IC50. Application of electroporation aimed to amplify the process of internalization of PS, thus enhancing its efficiency.

Usually, polysaccharides are big branched molecules (2 × 105–4 × 106 g/mol) 15, in many cases neutrally charged. We could speculate that the application of electrical pulses in the presence of polysaccharides may induce the reorganization of the cell surface and possible internalization into the cell membranes. The internalization of big molecules such as DNA into the cells was proved to be not through a single pore, but through the whole porated area 31, 32.

Both cell lines in suspension showed similar zeta‐potential values (MDA‐MB‐231: −22.99 ± 2.75 mV and MCF10A: −25.0 ± 0.87 mV) 33. Earlier studies of Zhang et al. 34 have shown data about the zeta‐potential value of MCF‐7 (breast low metastatic cancer cells) about −20.32 ± 2.43 mV and for MCF10A (normal cell lines) −31.16 ± 1.12 mV. The origin of negative surface charges on the cancer cells and normal cells is due to the different composition of their membranes. The cancer cells’ membrane is constituted by different negative phospholipids which give a negative charge to the outer leaflet of the membrane, whereas the surface of many non‐cancer cells is also composed by glycoconjugates which have ionogenic groups, such as sialic acid 35. The polysaccharides possess some anti‐adhesive properties and could reduce migration of neoplastic cells and thus decreasing the possibilities of proliferation 15. That is why one reason for the reduced viability could be connected with surface changing in signaling due to the reduced adhesion of cancer cells.

We continued our research with cell line MDA‐MB231 due to the highest cell viability reduction. Application of 75 µg/mL polysaccharide in combination with 200 V/cm electroporation induced 40% decrease in viability.

Cell morphology was followed at the 24th and 48th hour. Figure 6 reveals that control cells are with normal morphology (Figure 6A, D). Even after 24 h after the combined treatment of MDA‐MB231 cells with polysaccharide and electroporation, they became elongated, eventually due to some changes in the adhesion (Figure 6C).

Figure 6.

Figure 6

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Cell morphology of MDA‐MB231 cell line, (A) control after 24 h; (B) treatment with 75 µg/mL polysaccharide after 24 h; (C) combined treatment with 75 µg/mL polysaccharide and 200 V/cm after 24 h; (D) control after 48 h; (E) treatment with 75 µg/mL polysaccharide after 48 h; (F) combined treatment with 75 µg/mL polysaccharide and 200 V/cm after 48 h. Magnification 10x

To check if the reduced viability is due to DNA fragmentation (apoptosis), we isolated DNA from MDA‐MB231 cells. DNA fragmentation was not observed. We can conclude that cell death is not due to apoptosis.

4. CONCLUDING REMARKS

In this study we cultivated, isolated, and partially characterized the polysaccharide produced by the red alga Porphyridium sordidum. Sugar composition analysis by HPLC showed that the investigated sample was composed of Xyl:Glc and Gal:Man:Rha in a molar ratio of 1:0.52:0.44:0.31. The registered antitumor effect of the isolated polysaccharide is cell type specific. The electrical pulses additionally increase the cytotoxicity of neoplastic cells in the presence of polysaccharide, i.e. possess some adjuvant therapeutic effect. This mechanism resembles the reorganization, respectively entrance of other biopolymers molecules such as DNA. The effect is connected with the appearing morphological changes.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

ACKNOWLEDGMENT

This work was supported by Grant DN 11/2 of the Bulgarian National Science Fund.

Nikolova B, Semkova S, Tsoneva I, et al. Characterization and potential antitumor effect of a heteropolysaccharide produced by the red alga Porphyridium sordidum . Eng Life Sci. 2019;19:978–985. 10.1002/elsc.201900019

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