Carrageenan delays cell cycle progression in human cancer cells in vitro demonstrated by FUCCI imaging
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Carrageenan is a sulfated polysaccharide that exists in red seaweeds recently shown to have anticancer properties. Previous findings show various effects of carrageenan suppressing tumor cell growth. One of the hallmarks of cancer is uncontrolled proliferation, a consequence of loss of normal cell-cycle control, that underlies tumor growth. Recently there is an increasing interest in potential anticancer agents that affect cell cycle in cancer cells. Thus, in this study we investigated the effects of carrageenan on the tumor cell cycle.
Using human cervical carcinoma cells (HeLa) cells as and human umbilical vein endothelial cells (HUVEC), the cytotoxic effects of kappa carrageenan (k-CO) and lambda carrageenan (λ-CO) at the concentrations of 250–2500 μg/mL were observed. Cell viability was determined using the MTT assay while cell death rates were determined using staining with calcein-AM/propidium iodide. Cell-cycle profile and progression were demonstrated with HeLa cells expressing FUCCI (fluorescence ubiquitination-based cell-cycle indicator) probes (HeLa-FUCCI).
Carrageenan had no significant effect on HUVEC (normal cells). In contrast both forms of carrageenan were cytotoxic towards HeLa cells (cancer cells). Furthermore, according to cell-cycle analysis with FUCCI cells, the cell cycle of HeLa cells was delayed in specific phases due to different carrageenan treatments.
Considering these results, it could be suggested that carrageenan affects the cell-cycle of HeLa cells not only by arresting the cell cycle in specific phases but also by delaying the time needed for the cell to progress through the cell cycle. Additionally, different types of carrageenans have different effects on cell cycle progression. This effect of carrageenan towards cancer cells could possibly be developed into a tumor cell-specific anticancer agent.
KeywordsCarrageenan Algae Cancer Cell cycle Human cervical carcinoma cells Human umbilical vein endothelial cells Fluorescence ubiquitination-based cell-cycle indicator
Cancer is the leading cause of death worldwide, accounting for more 8.2 million deaths in recent years . Many polysaccharides have been isolated from mushrooms, fungi, yeast, algae, lichens and plants in the search for potential anticancer drugs. The biological activities of these polysaccharides have attracted considerable attention in the biotechnology and medical fields . In the search for novel compounds with antitumor properties, marine bioresources have become particular interest given their unique bioactivities . Marine algal cell walls were reported to contain sulfated polysaccharides, which are not found in land plants and may have specific functions in ionic regulation . Later studies revealed sulfated polysaccharides from marine algae have many biological and physiological activities including anticoagulant , antithrombotic , anti-inflammatory , antiviral , and activities .
A sulfated polysaccharide from algae that has been recently studied because of its interesting bioactivities is carrageenan. Carrageenan is a highly sulfated polysaccharide found in marine red algae of the family Rhodophyceae . Carrageenan is used as a stabilizer, gelling agent, thickener, binder and additive in various food and pharmaceutical industries. Carrageenans are composed of linear chains of D-galactopyranosyl units linked via alternated (1 → 3)-β-D-and (1 → 4)-α-D-glucoside , in which sugar units have one or two sulfate groups. From the commercial point of view, the most important carageenans can be categorized into kappa (k-), iota (i-), and lambda (λ-) carrageenans, which differ in the number and position of the sulfate groups. Analysis of their structures can be performed by acidic hydrolysis, for which methods have been developed based on reductive hydrolysis [12, 13, 14]. Additionally recent studiesshowthatcarrageenan, exhibits many biological and physiological activities besides its antitumor potential , including anticoagulant [16, 17], antithrombotic [18, 19], and anti-inflammatory properties [20, 21]. However, in the present study we would like to demonstrate carrageenan mechanism in affecting tumor cell cycle. Previous findings found that carrageenan has the potential to arrest the cell cycle in certain stages such as G2  or S phase . Many conventional anticancer treatments kill cells regardless of whether these cells are normal or cancerous. Based on the discovery that cell cycle characteristics of cancer cells are different compared to normal cells, potential antitumor agents that are able to affect the cell cycle could be a good target for antitumor drug research. Thus, we suggest it would be important to study how carrageenan affects the cell cycle of human cancer cells. In the present study we demonstrate for the first time cell cycle progression of effected human cancer cells by kappa carrageenan (k-CO) and lambda carrageenan (λ-CO) with fluorescence ubiquitination-based cell cycle indicator (FUCCI) that was developed by Sakaue-Sawano et al. . With FUCCI, we are able to observe cell-cycle progression in real time by measuring the expression of an orange-red fluorescent protein by G0/G1 cells and a yellow-green fluorescent protein by S/G2/M cells.
Cell culture and treatment
The cell line that was used in this experiment for cytotoxicity analysis was Human Carcinoma Cervical Cell lines (HeLa) for human tumor cell models and Human Umbilical Vein Endothelial Cells (HUVEC) for human normal cell models. Cell cycle progression was observed by HeLa cells (HeLa-FUCCI) expressing the FUCCI (fluorescent ubiquitiniation-based cell cycle indicator) probes provided by the RIKEN BioResource Centre (RIKEN BRC). HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium from Sigma-Aldrich supplemented with 10 % fetal bovine serum (FBS), at 37 °C in 5 % CO2 humidified atmosphere. HUVEC were cultured in Molecular, Cellular, and Developmental Biology (MCDB) 131 medium supplemented with 10 ng/mL epidermal growth factor, 10 μg/mL heparin and 10 % FBS.
HeLa and HeLa-FUCCI cells were seeded at a density of 2x105 cells/well in 96 well plates containing Dulbecco’s Modified Eagle’s Medium supplemented with 10 % FBS. HUVEC were cultured in MCDB 131 medium supplemented with 10 ng/mL epidermal growth factor, 10 μg/mL heparin and 10 % FBS. After overnight incubation, k-CO and λ-CO (purchased from Sigma-Aldrich) were added to each assay at concentrations of 250,500,1000, or 2500 μg/mL were added to each assay and maintained at 37 °C in a water saturated atmosphere containing 5 % CO2. Cells were incubated in the presence of k-CO and λ-CO for 72 h before cell count analysis.
Cell growth inhibtion assay
Manual cell count were also conducted using a hemocytometer and triplicate readings were recorded. Inhibitory activity expressed as the 50 % inhibition concentration (IC50) is the concentration of carrageenan that can inhibit cell growth by 50 % . The IC50 values were calculated using the following formula to determine the regression equation y = a + bx.
Quantification of live and dead cells
To quantify the ratio of live/dead cells, cells were stained with the fluorescent probes calcein AM and propidium iodide (PI). Because of the permeable ability of the cell membrane, calcein AM was used to stain viable cells, while PI was used to label dead cells HeLa and HUVEC were plated onto 35 mm2 glass-bottom culture dishes and cultured overnight to achieve cell adhesion. Then cells were treated with different concentrations (250,500,1000, or 2500 μg/mL) of K-CO and λ-CO. After 72 h treatment, the cells were washed three times with a PBS, followed by incubation with a PBS solution mixed with 2 μL calcein AM at 37 °C for 15 min. Finally, cells were stained with 2 μL PI before visualization under. BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). The dead cell ratio was calculated using Image J software .
Cell cycle profile analysis
Cell cycle profiles of the cells following treatment with k-CO and λ-CO were observed using HeLa-FUCCI cells over72h. FUCCI was developed to visualize the dynamics of cell cycle progression. HeLa-FUCCI cells express two fusion proteins : monomeric Kusabira Orange 2 (mKO2) fused to amino acids 30–120 of Cdt1, and monomeric Azami Green fused to amino acids 1–110 of Geminin. This combination of fused proteins causes cells to emit red fluorescence at the early G1 phase immediately after mitosis, and emit green fluorescence in S/G2/M phases. After 72 h treatment of carrageenan on HeLa-FUCCI cells cultured in 35 mm2 glass bottom dishes, scanning microscopy was performed with the Bz-9000 fluorescent microscope (Keyence). Scanning and image acquisition were controlled using Bz-II analyzer software (Keyence). The tracing data were imported to Image J for quantification of cells emitting fluorescence of each cell cycle phase.
For a more accurate observation of cell cycle progression due to carrageenan treatment, we set up time-lapse imaging observation with 10 min interval for 72 h. HeLa-FUCCI cell cycle progress was captured using a BIOREVO BZ-9000 fluorescence microscope (Keyence). During imaging, cells were cultured in 35 mm2 glass-bottom dishes, held in an incubation chamber to maintain temperature at 37 °C with humidified atmosphere containing 95 % air/5 % CO2. Average time of cells from six independent observations to complete cell-cycle phases were calculated for cell-cycle time estimation.
Effects of k- and λ- carrageenan on cell growth of normal human cells in vitro
Effects of K- and λ- carrageenan on cell growth of human cancer cells cells in vitro
Inhibition carrageenan concentrations causing 50 % cell death (IC50) of 72 htreatment
550.8 ± 7.6
475 ± 12
Viability staining assay
Viability staining was necessary for further confirmation of the current result. Viability staining is an essential tool to obtain a clear picture of cell viability towards treatment of potential drugs. The cytotoxic properties of k-CO and λ-CO were investigated further using a double-labeling procedure with the fluorescence markers calcein-AM and PI, which simultaneously stain live and dead cells in green and red, respectively. Intracellular esterases in live cells can convert the virtually nonfluorescentcell-permeable calcein-AM into the intensely fluorescent calcein. PI enters cells with damaged membranes and enhances fluorescence by binding to DNA, thereby producing a bright red fluorescence in dead cells . HeLa cells were treated with k- and λ- carrageenan at concentrations of 250–2500 μg/mL for 72 h then loaded with calcein-AM and PI before observation with fluorescence microscopy. The population of live (calcein-positive) and dead (PI-positive) cells can be easily differentiated.
Live cell imaging of cell cycle progress
Time required for cells to undergo phases of cell cycle in treatment of k-CO and λ-CO (1000 μg/mL). Data presented are means of ± SD, n = 6 independent observations
Time length (h)
1.5 ± 0.7
9.3 ± 1.8
1.7 ± 0.8
14.1 ± 1.9
26.6 ± 0.6
1.0 ± 1.1
16.8 ± 4.0
2.2 ± 1.2
30.1 ± 7.3
50.2 ± 2.9
1.3 ± 0.4
26.5 ± 4.9
2.5 ± 0.2
32.5 ± 5.1
59 ± 4.6
Carrageenan is a polysaccharide that exists in the cell walls of marine red algae and is widely used in studies concerned with its antitumor and cytotoxic activities . Previous findings show carrageenan as a potential antitumor agent [28, 29, 30]. Considering one of the hallmarks of cancer is uncontrolled proliferation, a consequence of the loss of normal cell-cycle control, there has been a. increasing interest in potential anticancer agents that affect the cell-cycles of cancer cells . Thus, in this study we investigated how carrageenan affects tumor cell cycle.
In this study we demonstrated cytotoxic effects of carrageenan towards cell cycle of human cancer cells in HeLa expressing FUCCI probes . Two types of carrageenan, kappa (k-CO) and lambda (λ-CO) carrageenan were used because sulfate contents vary in each type of carrageenan . These sulfated moieties in saccharides are believed to play an important role in manifestation of beneficial bioactivity . Thus cytotoxic properties of λ-CO which has more sulfate groups might differ from k-CO. Cytotoxic effects are generally considered as effects of a compound resulting in cell damage or death . Our current results show dissimilar properties of k-COand λ-CO in effecting cell proliferation, cell death ratio, and cell cycle progression.
In vitro cytotoxicity tests are mainly performed to screen potentially toxic compounds that affect basic molecular functions such as cell growth. For acute toxicity measurement, 3 h of exposure is sufficient to reulst in toxic effects in the cell . In the case of measuring cell proliferation, the time of exposure should be 72 h, because that amount of time is required to obtain about three cell divisions . The HeLa cell line chosen as models for this investigation is an immortal cell line generally used in toxicity tests. The ability of this cell line to proliferate rapidly makes it possible to conduct several experiments aimed mainly at detection of biological activity of test substances and also provide long-term culture . The cytotoxic effects of compounds can be classified by the IC50 values. IC50 values less than 100 μg/mL indicate a potentially cytotoxic compound. IC50 values in the range of 100–1000 μg/mL are considered to correspond with moderate cytotoxic effects, and compounds with IC50 values greater than 1000 μg/mL are considered non-toxic to the cells [38, 39]. In this study, k-CO and λ-CO both were found to cause moderate toxic effects on HeLa cells because IC50 values were in the range of 100–1000 μg/mL (Table 2). However λ-CO potentially possesses stronger cytotoxic effect compared with k-CO based on IC50 values. λ-CO was reported to have higher sulfate content than k-CO which results in stronger antioxidant activity. Furthermore, high antioxidant activity may potentially result in anti-proliferative effects . However, further studies are needed to confirm the correlation between sulfate content and anti-proliferative properties. Live:dead cell assays were performed to examine cell death through determination of intracellular esterase activity and plasma membrane integrity. The data showed k-CO resulted in a higher ratio of dead cells after staining with calcein-AM and PI. However, images of HeLa cells treated with λ-CO displayed lower cellular confluence implying cellular proliferation is suppressed by the anti-proliferative potential of λ-CO. The findings of the current study are consistent with those of other studies that showed cell death induced via cellular toxicity caused by carrageenan treatment in human tumor cells . To determine the cytotoxic effects of carrageenan on human normal cells, we used HUVEC. Both k-CO and λ-CO showed no significant cytotoxic effects towards HUVEC viability. We expected cancer cells to be generally resistant to apoptosis compared with normal human cells. However, as seen in responses to potential anticancer agents in cell cultures, most cancer cell lines are more sensitive to apoptosis compared with normal cell lines [42, 43].
Cell cycle arrest and cell death are two important mechanisms involved in anti-cancer drug development. Uncontrolled cellular proliferation is a general characteristic of all cancer cells, and the blockade of the cell cycle is regarded as an effective strategy for eliminating cancer cells. Many chemotherapeutic agents have shown anti-proliferative effects via arresting cell division at certain checkpoints in the cell cycle. The concept of cell cycle-mediated cell death has gained increasing attention, as targeting this pathway may provide an opportunity to overcome drug resistance, decrease mutagenesis and reduce toxicity.
Our study findings suggest that carrageenan significantly inhibits HeLa growth not only by arresting the cell cycle in specific phases but also by causing a delay in cell cycle progress. Furthermore, both λ-CO and k-CO have different effects on the cell cycle in HeLa cells. k-CO was seen to delay the cell cycle in G2/M phase while λ-CO stalled the cell cycle in both G1 and G2/M phase, resulting in a longer cell cycle compared with either k-CO treated and untreated cells. Furthermore, λ-CO displayed suppression for the ability of the cell to divide, demonstrating a strong anti-proliferative effect. In contrast, carrageenan shows no significant effects towards HUVEC. However, in vivo studies and trials on other human cancer and normal cell lines are needed to confirm these results for more advanced development of carrageenan as a potential anticancer agent.
%, percentage; μg/mL, microgram/milliliter; Abs, Absorbance; Calcein-AM/PI, calcein-acetoxymethyl diacetylester/propidium iodide; DMEM, Dulbecco’s Modified Eagle’s Medium; EGF, epidermal growth factor; FBS, fetal bovine serum; FUCCI, fluorescence ubiquitination-based cell cycle indicator; h, hours; HeLa, human carcinoma cervical cell lines; HUVEC, human umbilical vein endothelial cells; IC50, inhibitory concentration of 50 %; mAG, monomeric azami green; MCDB, molecular, cellular, and developmental biology; mKO2; mm2, square millimeter; monomeric kusabira orange 2; MTT, methyl thiazolyl tetrazolium; ng/mL, nanogram/milliliter; nm, nanometer; °C, degree celcius; RIKEN BRC, RIKEN BioResource Centre; k-CO, kappa carrageenan; λ-CO, lambda carrageenan
The author wish to express their gratitude to JSPS (Japan Society for the Promotion of Science) for the grant awarded (Grant-in-Aid for Scientific Research/C/No. 26460299) and Fukushima Medical University.
JSPS (Japan Society for the Promotion of Science) for the grant awarded (Grant-in-Aid for Scientific Research/C/No. 26460299) and Fukushima Medical University.
Availability of data and materials
All the data is contained in the manuscript.
ESP, MM, KD, and AH involved in the experimental design and progress supervision. ESP carried out the experiments and collected the data. ESP wrote the manuscript under the supervision of MM, KD, and AH; all authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
“Not applicable” in this section.
Ethics approval and consent to participate
“Not applicable” in this section.
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