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Protistology 18 (1): 60-71 (2024) | doi:10.21685/1680-0826-2024-18-1-6
Protistology
Original article
The effect of biologically active compounds of dinoflagellates Prorocentrum cordatum on proliferation and motility of the transformed CT26 cells
Natalia A. Filatova, Sergei O. Skarlato and Sofia A. Pechkovskaya*
Institute of Cytology, Russian Academy of Sciences, Saint Petersburg 194064, Russia
| Submitted January 11, 2024 | Accepted March 16, 2024 |
Summary
Marine dinoflagellates produce a large number of biologically active compounds (BAC), including unique pigments and secondary metabolites of various chemical composition. In this study, we assessed the biological activity of ethanol (EtE) pigment-containing and aqueous (AqE) protein-containing extracts obtained from the marine dinoflagellate Prorocentrum cordatum. BAC can influence the growth and development of marine ecosystems through a process known as allelopathy, and of the greatest interest is studying their toxicity and impact on the proliferation, cell cycle, and motility of the target cells. To investigate the BAC effects on the aforementioned characteristics, we selected the CT26 transformed cell line for model experiments allowing us to evaluate the antitumor activity of the obtained extracts containing BAC. Colorimetric analysis using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) demonstrated a significant decrease in the viability of CT26 cells upon treatment with AqE, but not with EtE at the selected concentrations. The IC50 values of EtE and AqE were 374.9 and 2.4 ^g of dry weight/ml, respectively. Flow cytometry analysis showed that treatment with EtE affected the cell cycle distribution, leading to cells retention in the S phase. Neither AqE nor EtE treatments induced apoptosis. Using the wound healing assay, we found that both EtE and AqE inhibited the motility of CT26 cells by 1.4—1.8 times depending on the time after exposure. These results suggest that P. cordatum can be a source of valuable biologically active compounds that inhibit proliferation and reduce the motility of CT26 cells.
Key words: Dinoflagellates, Prorocentrum cordatum, CT26, bioactive compounds, antitumor activity, cell motility
https://doi.org/10.21685/1680-0826-2024-18-1-6
Corresponding author: Sofia A. Pechkovskaya. Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, 194064 St. Petersburg, Russia; sapechkovskaya@gmail.com
© 2024 The Author(s)
Protistology © 2024 Protozoological Society Affiliated with RAS
Introduction
Dinoilagellates are among the main primary producers in aquatic ecosystems and are capable of synthesizing a wide range of complex organic substances ofvarious chemical composition, serving as a source of novel biologically active compounds (BAC) (Cousseau et al., 2020; Ferrara, 2020). Over the last 20 years, there has been a significant increase in the search for and identification of BAC from marine organisms with various unique properties that allowed using them in pharmacology, biomedicine and toxicology. It has already been shown that algae are able to synthesize substances with antiviral (Matsuhiro et al., 2005), antibacterial (Najdenski et al., 2013), antifungal (Li et al., 2006) and antitumor (Murphy et al., 2014) properties.
The dinoflagellate Prorocentrum cordatum is widely used as a planktonic model species, which possesses a broad ecological niche due to high adaptability to variable environmental conditions, and demonstrates a pronounced invasive potential (Telesh et al., 2016, 2024; Telesh and Skarlato, 2022). The successful survival of these protists, their effective range expansion in new marine areas, and the bloom formation capacities may be partly related, among other factors, to the production of a number of unique secondary metabolites and pigments, such as peridinin, diadinoxanthin, diatoxanthin, etc. (Zapata et al., 2012; Beedessee et al., 2020). In the recent years, dinoflagellates of the genus Prorocentrum have been found to harbor a large number ofunique secondary metabolites, including belizentrin (Dominguez et al., 2014), prophocentrolides (Amar et al., 2018; Lee et al.,
2019), formosalides (Lu et al., 2009), prophocentrol (Sugahara et al., 2011), and others. While their pharmacological potential currently is still under study, the anticancer properties of prophocentrolides have already been demonstrated (Lee et al.,
2020).
Secondary metabolites are released by the dinoflagellate cells into the surrounding environment, where they can positively or negatively affect the growth of other organisms and, consequently, influence the development of aquatic communities and ecosystems (Suikkanen et al., 2011; Hakanen et al., 2014). This widespread phenomenon, known as allelopathy, can be caused by many primary producers (Inderjit and Dakshini, 1994) and it is assumed to play an important role in the successful development of microalgae and the formation of blooms (Takamo et al., 2003; Granéli et al., 2008).
Recently, there has been a global surge in the screening ofbiologically active compounds produced by dinoilagellates, and pharmacological properties of the dinoflagellate toxins have been demonstrated. Specifically, due to the ability to influence the variety of biological receptors and metabolic processes, saxitoxins and tetrodotoxins were evidenced to possess analgesic and antimicrobial effects (Borowitzka, 1995; Epstein-Barash, 2009; Berde et al., 2011). Other unique bioactive compounds have also been identified in dinoflagellates; however, much more data on other dinoflagellate species would be required to evaluate the pharmacological potential of these compounds (Assun?ao et al., 2017; Cousseau et al., 2020).
Currently, approximately 60% of drugs used in hematology and oncology are natural products or their derivatives (Dyshlovoy and Honecker, 2018). It is known that 90% of cancer patient deaths occur because of metastasis. Therefore, the most relevant approach is to search for compounds with antitumor and migrastatic effects aimed at inhibiting the mo-tility of tumor cells and reducing their ability to migrate. Another important goal is to identify natural compounds that would be least toxic to patients.
However, there is relatively little information in the modern scientific literature concerning dino-flagellate-produced molecules with antitumor acti-vity; in most cases, the effect of total extracts is being investigated. For instance, the chloroform fraction of Amphidinium carterae extracts was shown to be most active against HL-60 cells, reducing their viability by about 50% after 24 h exposure at a concentration of 50 ^g/ml (Samarakoon et al., 2013). Methanol extracts obtained from different types of dinoflagellates have been found to exhibit a cytotoxic effect against HL-60 cancer cells, resulting in a decrease in cell viability from 40 to 60% compared to the control at a concentration of 50 ^g/ml (Shah et al., 2014). Additionally, a water-soluble high-molecular compound isolated from a toxic marine dinoflagellate, Alexandrium minutum, activates the synthesis of genes involved in specific cell death in human lung adenocarcinoma (Galasso et al., 2018). The impact of extracts from microalgae on the motility of tumor cells of different etiologies has been also demonstrated (Somasekharan et al., 2016; Suh et al., 2017; Alateyah et al., 2022). However, little is known about the effect of extracts from dinoflagellate cells on the ability of cancer cells to meta-stasize.
Generally, the active substance of the extracts and their chemical structures remain unknown.
Some studies indicate that the pigment peridinin possess pharmacological and antitumor potential (Sugawara et al., 2007; Yoshida et al., 2007). Thus, dinoflagellate metabolites demonstrate an impressive range of biological effects, including antimicrobial and antitumor properties, making them a promising source of antitumor compounds. The goal of our study was to obtain extracts with an inhibitory effect on the motility oftransformed CT26 cells without exhibiting high toxicity. To achieve this, a protocol for the isolation of extracts from cells of the dinoflagellate P. cordatum was developed, and studies of the effect of these extracts on survival, proliferative activity, apoptosis and motility of CT26 target cells in the in vitro experiments was performed.
Material and methods
eliminate cell debris, and the resulting supernatant was collected. AqE and EtE were then evaporated to dryness using a concentrator (Martin Christ RVC-33IR, Martin Christ, Germany). One milligram of the dried algal powder was extracted with 100 ^l of ethanol or PBS. All concentrations are expressed in micrograms of dry weight.
The analysis of the absorption spectrum for total pigment extracts from P. cordatum cells was conducted using the Nanophotometer NP80 (Implen, Munich, Germany) at room temperature, covering a wavelength range of 350—750 nm. To quantify the total chlorophyll a content in ethanol, the equation proposed by Ritchie (2006) for the dinoflagellate species was applied: chl a (^g/ml) a -2.6094 x A629 + 12.4380 x A665.
For AqE, the protein concentrations were determined by employing the standard Bradford assay at 595 nm.
MICROALGAE CULTURE
CELL CULTURE
The strain CCAP 1136/16 of the dinoflagellate Prorocentrum cordatum Dodge, 1975 (major synonym: Prorocentrum minimum (Pavillard) Schiller, 1933) was acquired from the Culture Collection of Algae and Protozoa at the Scottish Marine Institute in Oban, UK, and used for the experiments. The cells were cultured in an f/2 medium (Guillard and Ryther, 1962) based on artificial seawater, devoid of silicate, at salinity of 25 PSU, temperature of 20 °C, irradiance of 50 ^mol photons m-2 s-1 and a 12 h light: 12h dark cycle. Sterilization of artificial seawater and all stock solutions was achieved through autoclaving or sterile filtration.
Preparation and characterization of algal extracts
P. cordatum cells were collected by centrifuga-tion at 1500 g for 10 min at 20 °C. After carefully decanting the supernatants, tubes containing cell pellets were frozen and stored at —80 °C. Before extraction, the cells were thawed, resuspended, and homogenized in either 96% EtOH (for ethanol extracts, EtE) or PBS (for aqueous extracts, AqE). Cell disruption was achieved using a FastPrep TM-24 homogenizer (MP Biomedicals, USA) with lysing matrix F (MP Biomedicals, USA) at a stirring rate of 6 m/s. For ethanol extraction, cells were incubated at 4 °C in the dark for 48 h. Subsequently, both ethanol and aqueous extracts underwent centrifugation at 10000 g for 10 min to
Cell lines CT26 (Mus Musculus colon carcinoma) were obtained from the American Type Culture Collection (ATCC CRL-2638; Manassas, VA, USA). Cell lines were routinely maintained in RPMI 1640 medium (HyClone, USA) containing 10% FBS (HyClone, USA), 100 ^M of streptomycin, and 100 U/mL of penicillin in an atmosphere of 5% CO2 at 37 °C.
Cell viability assay
The extracts' inhibitory effect on cell growth against CT26 cell lines was evaluated through the MTS assay. Cells were initially plated in 96-well plates at a density of 5x 103 cells per well. After a 24 h incubation, the cells were treated with EtE at concentrations of 20, 40, and 90 ^g/mL, while AqE was applied at concentrations of 3, 6, and 9 ^g/mL. The cells were then further incubated for additional 24 h. To determine cell proliferation, MTS reagent (Promega, USA) was introduced into each well (20 ^L), and the plates were incubated for 2 h in a 5% CO2 atmosphere at 37 °C. The resulting absorbance was measured at 495 nm using a plate spectrophotometer (Multiskan FC, Thermo Fisher Scientific, USA).
APOPTOSIS
For apoptosis analysis, cells were seeded into 24-well plates at a density of 5x 104 cells per well.
After 24 h of incubation, cells were treated with a medium containing aqueous or ethanol extracts at a concentration of 0.3 ^g/mL. Subsequently, cells were washed, collected by trypsinization, and stained with Annexin V-FITC and propidium iodide (Sigma, USA) following the manufacturer's protocol. Cellular apoptosis was assessed using flow cytometry (EPICS XL, Beckman Coulter, USA).
Cell cycle
The cell cycle distribution, encompassing the G0/G1, S, and G2/M phases, was evaluated by assessing the relative DNA content in cells and analyzed using flow cytometry. For the cell cycle analysis, the samples underwent three washes with phosphate-buffered saline (PBS) followed by staining for DNA content using 0.2 mg/mL saponin (Sigma, USA), 0.25 mg/mL RNase (Sigma, USA), and 0.05 mg/mL propidium iodide (Invitrogen, USA). After a 30 min incubation at room temperature, the samples were analyzed using a flow cytometer EPICS XL (Beckman Coulter, USA). Data analysis was performed using ModFit LT software (Verity Software House, USA). The results section provides the average values along with their variations based on the obtained data, and illustrative diagrams with corresponding values are presented.
Wound healing assay (Scratch test)
MH22a cells were plated in 35 mm Petri dishes at a concentration of 5 x 105 cells per dish and allowed to reach confluence. Scratch wounds were created using a 10 ^L pipette tip, and detached cells were eliminated by washing with PBS. To impede cell proliferation, the culture medium was replaced with serum-free DMEM. Extracts were introduced into the cultures at concentrations of 40 ^g/mL for EtE and 3 ^g/mL for AqE, followed by a 48 h incubation period. Microscopic images were captured using an AxioVert microscope (Carl Zeiss Microimaging GmbH, Jena, Germany). The NIH ImageJ software was employed to calculate the percentage of wound closure in 10 randomly selected fields.
Statistical analysis
The statistical analysis was conducted utilizing MaxStat 3.06 software (MaxStat Software, Germany). Means ± standard error (mean ± SE) were calculated based on data from three independent
Fig. 1. Absorption spectrum of the ethanol extract from P. cordatum at room temperature. The figure shows the absorption values that were used to calculate the Chl a content in the samples.
experiments and compared using either Student's t-test or the nonparametric U-Wilcoxon-Mann-Whitney test. Statistical significance among groups was determined at a threshold of p < 0.05.
Results and discussion
Characteristics of ethanol (EtE) pigment-containing AND AQUEOUS (AqE) PROTEIN-CONTAINING EXTRACTS FROM PROROCENTRUM CORDATUM
The EtE enriched with P. cordatum pigments, such as peridinin, chlorophyll a and c, diadinoxan-thin, diatoxanthin, etc. were obtained from the dinoflagellate strain CCAP 1136/16. To analyze these extracts and determine the total Chl a content, we measured the absorption spectrum of samples at room temperature. The spectrum exhibits a wide band spanning from 400 to 550 nm with two clear peaks at 469 and 677 nm. The Chl a Soret band at 438 nm overlaps with the broad band created by peridinin and other carotenoids. The average Chl a concentrations were calculated based on the absorption values at 629 nm and 665 nm and using the appropriate coefficients for ethanol solutions (Ritchie, 2006) (Fig. 1). The average Chl a content within the samples was determined to be 0.1 ± 0.002 ^g per 1 mg of dry weight.
The obtained spectra correspond to those reported in the literature (Johnsen et al., 1994; Jiang et al., 2012). Previous studies have demonstrated the crucial role of pigments in inhibiting the growth and proliferation of tumor cells. For instance, ethanol extracts obtained from peridinin-containing di-
Fig. 2. Effect of different concentration of ethanol (EtE) and aqueous (AqE) extracts on growth of CT26 cells. The bar charts display the proportions of living and dead cells compared to the control ± SEM (n = 3). The asterisk (*) denotes a significant difference between the control and experimental groups, p < 0.05.
noflagellates Heterocapsa triquetra exhibited the capacity to impede the growth of A2058 cells, showing moderate antiproliferative effects (25.6— 34.5% growth inhibition). Fractionation of these extracts revealed 11 colored fractions and a single colorless fraction. Among the pigments identified, chlorophyll c2 exhibited the highest activity, but peridinin, dinoxanthin, and diatoxanthin have also demonstrated the inhibitory effect towards target cells (Haguet et al., 2017). Although information regarding peridinin is still scarce, there is significant evidence of the pharmacological effects of other microalgae carotenoids, such as fucoxanthin (found in fucoxanthin-containing dinoflagellates and diatoms) and astaxanthin (a red carotenoid from Chlorophyta). These carotenoids demonstrate various anticancer properties, including anti-proli-ferative effects, induction of cell cycle arrest, pro-
motion of apoptosis, and inhibition of metastasis (Shao et al., 2016; Ahmed et al., 2022).
In the AqE extracts, the protein concentration was determined using the Bradford method, resulting in a concentration of 94.8 ± 2.8 ^g per 1 mg of dry weight. There is limited information on the cytotoxic effects of aqueous extracts from marine microalgae and cyanobacteria on tumor cell cultures. However, Galasso and colleagues (Galasso et al., 2018) identified a 20 kDa glycopeptide isolated from a water-soluble fraction of the extract of the dinoflagellate Alexandrium minutum, which showed cytotoxic effects against A549 cells. Additionally, aqueous extracts from different cyanobacteria species were found to inhibit the growth of cell cultures like EACC and HepG2, with IC50 concentrations ranging from 1.36 to 3.93 mg/100 g (Shanab et al., 2012).
Effect of extracts on cell viability
For this study, we selected transformed cell line CT26 with a high metastatic potential, which can be implanted into Balb/c mice. The viability of CT26 cells was assessed at 24, 48 and 72 h after treatment with extracts using the MTS assay. The treatment with EtE did not lead to a significant decrease in the number of living cells. Alternatively, the treatment with AqE after 48 h showed a significant decrease in living cells number starting at a concentration of 3 ^g/ml (Fig. 2). The IC50 values of EtE and AqE were 374.9+2.5 and 2.4+0.1 ^g of dry weight/ml, respectively.
The in vitro and in vivo inhibition of CT26 cell growth by extracts from various plants has been thoroughly studied (Kim et al., 2019; Hosseinzadeh et al., 2020; Makaremi et al., 2021). However, information about the action of substances isolated from seaweed on CT26 cells is extremely scarce, and information about extracts from microalgae is practically absent. For instance, it is known that the hydroalcoholic extract of red seaweed Sargassum oligocystum showed toxic activity and inhibited growth against CRC cells (Al-Aadily et al., 2022). Regarding unicellular algae, single studies indicate that the cell membrane fraction obtained from the green algae Chlorella sorokiniana inhibits the growth ofCT26 cells in both 2D culture and a spheroid assay (Ishigiro et al., 2017).
Effect of EtE and AqE on apoptosis
The number ofnecrotic and apoptotic cells after the EtE and AqE treatment showed no significant change compared to the control. The analysis revealed that the number of necrotic cells in all experimental groups did not exceed 4.6+0.2%, while the number of apoptotic cells reached 11.0+0.8% (Fig. 3).
Despite the absence of apoptosis induction in our experiments, it was previously demonstrated that aqueous extracts from marine algae Codium fragile induced apoptosis by reducing Bcl-xL levels and activating caspases 3 and 7 (Kim et al., 2008). Subsequently, it was shown that aqueous extracts and, in particular, sulfated polysaccharides from brown algae Ecklonia cava demonstrated cytotoxicity, apoptosis activation, suppression of migration ability, and enhancement of tumor suppression in cultured CT26 cells or CT26 tumors in BALB/cKorl syngeneic mice (Ahn et al., 2015; Gong et al., 2023).
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nil
EtE control EtE AqE control AqE ■ necrotic »apoptotic□ living cells
Fig. 3. Effect of ethanol (EtE) and aqueous (AqE) extracts on the apoptosis of CT26 cells. The bar charts display the mean percentage of living, necrotic and apoptotic fractions of cells in relation to the control (n =3).
Effect of extracts on cell cycle
The flow cytometry analysis revealed that EtE treatment resulted in a significant increase of cell numbers in the S phase and their decrease in the G2M phase (Fig. 4). Thereby, EtE not only demonstrated low toxicity to CT26 cells, but also led to an accumulation of cells at the boundary between the S and G2M phases. However, the AqE treatment did not cause any significant cell cycle alterations.
Despite a large array of data regarding the effect of plant extracts on the cell cycle of CT26 tumor cells, the antitumor properties of algae BACs have been scarcely studied. For instance, previous research demonstrated that carob leaf polyphenols induced G/S cell cycle arrest in CT26 cells, possibly resulting from an increase in cyclin p27 expression (Ghanemi et al., 2017). Additionally, there is evidence that imbricatolic acid, isolated from the me-thanolic extract of Juniperus communis berries, induces the accumulation ofG: phase cells and down-regulates cyclins A, D1, and E1 in CaLu-6 cells (Lai et al., 2021).
Effect of extracts on cell motility
Tumor cell motility was assessed by the wound healing assay. We found that 72 h after EtE treatment, the CT26 cells' ability to fill the damaged area decreased by 2.5 times compared to the control, resulting in an area-filling rate of 26.9+2.7%. In contrast, the treatment with AqE did not lead to any significant changes in the cell's motility, with the damaged area being filled to 68.2+6.1% (Fig. 5).
Fig. 4. Effect of different concentrations of ethanol (EtE) (B) and aqueous (AqE) (C) extracts on the distribution ofCT26 cells in the cell cycle. A. The most typical histograms of distribution of cells in the cell cycle are presented. The bar charts display the average distribution of the cells across the cell cycle ± SEM (n =3). The asterisk (*) denotes a significant difference between the control and experimental groups, p < 0.05.
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Fig. 5. Effect of ethanol extract (EtE) and aqueous extract (AqE) on CT26 cell migration evaluated through the wound-healing migration assay. A — Representative images from two separate experiments performed in triplicate (magnification x40); B — the percentage of cell-covered area at 24 h and 48 h relative to 0 h is depicted as the mean ± SEM (n=3). The asterisk (*) denotes a significant difference between the control and experimental groups, p < 0.05.
Generally, substances that are non-toxic to cells and do not affect other parameters are considered as potential antistatic agents. Previously, it was evidenced that total extracts of fungi and plants are able to influence the motility of CT26 cells. Thus, it was established that the hot water and microwaved 50% ethanol extracts of Hericium erinaceus mushrooms effectively inhibited the proliferation and invasion of CT-26 colon carcinoma cells, as well as the metastasis and invasion of CT-26 cells to the lungs (Kim et al., 2013). Additionally, Panax ginseng extract was found to inhibit the adhesion, migration, and invasion of CT26 cells, as well as its lung metastasis through the regulation of epithelial-mesenchymal transition (EMT) via the Smad2/3-and p38/ERK signaling pathways (Kee et al., 2019).
Conclusion
in this study, we have developed the effective protocols for obtaining ethanol and aqueous extracts from the dinoflagellates P. cordatum. It was found that both AqE and EtE extracts affected cell motility, inhibiting it by 1.4—1.8 times depending on the time after exposure. At the same time, both extracts did not induce apoptosis. However, it was shown that pigment-containing EtE demonstrated significantly lower toxicity towards target cells compared to protein-containing AqE; moreover, EtE exhibited a cytostatic effect. This allows considering EtE as a more promising extract from P. cordatum for further research in identifying biologically active substances aimed at the development of preparations with a migrastatic effect, compared to AqE.
Acknowledgements
We are grateful to N.A. Knyazev for participating in the experimental part of the study. We thank A.G. Mittenberg and Y.A. Nashchekina for providing access to the facilities for extract evaporation. This research was supported by the Russian Science Foundation (project # 22-14-00056; https://rscf. ru/project/22-14-00056/). The use of equipment was partly financed by the Budgetary program # FMFU-2024-0012 at the Institute ofCytology RAS.
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