SUPPLEMENTARY MATERIAL
Polysaccharide extracts of the brown alga Sargassum asperifolium possess in vitro cancer
chemopreventive properties
Eman Mahmoud Raafat a, Amira M. Gamal-Eldeen b,c,*, Enas A. El-Hussieny d,
Eman F. Ahmed e, Amany Ali Eissa a
a
Department of Pharmacology and Toxicology, faculty of Pharmacy, Helwan university, Cairo, Egypt.
b
Cancer biology laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki,
12622, Cairo, Egypt.
c
Biochemistry Department, National Research Center, Cairo, Egypt
d
Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt
e
Department of Natural and Microbial Products Chemistry, National Research Center, Cairo, Egypt
* Corresponding author, A.M. Gamal-Eldeen: Fax: +20233370931, E.mail: aeldeen7@yahoo.com.
Abstract
The cancer chemopreventive activity of the polysaccharide extracts (E1-E4) of Sargassum asperifolium, a
brown alga in red sea shores in Egypt, was investigated. Tumor anti-initiation activity (the modulation of
carcinogen metabolism) indicated that E3 and E4 were potent anti-initiators by inhibiting the carcinogen
activator cytochrome P450-1A, and enhancing carcinogen detoxification enzymes GST. Only E4 significantly
enhanced quinone reductase activity. All polysaccharide extracts possessed anti-promotion property by their
anti-inflammatory activity. E3 and E4 dramatically induced the growth of spleen macrophages. E2, E3 and E4
significantly inhibited nitric oxide generation from lipopolysaccharide (LPS)-stimulated macrophages, while E1,
E3 and E4 led to significant inhibition of LPS-induced TNF-α. The extracts E1, E2 and E4 showed cytotoxicity
against HepG2 cells, where E2 and E4 induced cell death due to apoptosis. In conclusion, E3 and E4 are
promising cancer chemopreventive extracts, since they had tumor anti-initiating activity via their protective
modulation of carcinogen metabolism.
Key words: Sargassum asperifolium, cytochrome P450 1A, Cancer chemoprevention, Anti-initiating, Antipromoting, Apoptosis
Experimental
Extraction of polysaccharides from S. asperifolium
S. asperifolium was washed several times with distilled water, air dried at room temperature, grounded into fine
particles, and then sieved. The water soluble polysaccharides were extracted from S. asperifolium according to
[Zhuang et al., 1995], with some modifications. The powdered algal sample (510 g) was extracted repeatedly
with 80% ethanol at 80oC, to eliminate the low molecular weight ingredients. The resulted dried residual
materials were extracted three times with boiling distilled water at 100ºC for 4h. The filtrate was concentrated
and precipitated with 3% aqueous cetyl pyridinium chloride, and then centrifuged for 15 min at 3000 x g. The
precipitate was dissolved in 4M NaCl for 20 h at 40°C and re-precipitated again with 99% ethanol and the
precipitate (E1) (25.6 g) was dissolved in water, dialyzed and lyophilized. The precipitate was dissolved in 4M
NaCl for 20 hour at 40°C and re-precipitated with 99.8% ethanol. The residual dried alga was dissolved in
water, dialyzed and lyophilized (E2) (1.11 g). The dried residue resulted from the hot water extraction was
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weighted and extracted with 0.1M HCl at 95°C for 4h, repeatedly for three times. After filtration, the filtrate was
concentrated under reduced pressure and precipitated with 3% cetyl pyridinium chloride, the filtrate was
concentrated and precipitated with 99% ethanol. The precipitate (E3) (1.08 g) was dissolved in water, dialyzed
and lyophilized. The dried residue resulted from the filtration was weighted and re-extracted with 1.25 M NaOH
at 28°C for 24h for two times and then filtered. The residue was discarded and the filtrate was concentrated
under reduced pressure, adjusted to pH6 with 99% acetic acid, and then precipitated with 99% ethanol to give
(E4) (2.08 g). Finally, extracts E1, E2, E3, and E4 were lyophilized, and the powdered extracts were tested for
their cancer chemopreventive activities. A scheme for the extraction of polysaccharides from S. asperifolium
was illustrated in (Fig. 1).
Cell culture
Human hepatocarcinoma (HepG2) and mouse hepatoma (Hepa1c1c7) were purchased from ATCC, USA. HepG2 cells and Hepa1c1c7 cells were routinely cultured in Dulbeco’s Modified Eagle’s Medium (DMEM)
supplemented with 100 U/ml streptomycin sulfate, 250 ng/ml amphotericin B, 2 mM L-glutamine, 100 U/ml
penicillin G sodium, and 10% fetal bovine serum (FBS). Cells were cultured at 37 C in humidified air containing
5% CO2. Cells were harvested by trypsin/EDTA. All experiments were repeated four times, and the data was
represented as (Mean ± SEM). All cell culture material was obtained from (Gibco® /Invitrogen), while all other
chemicals were from Sigma–Aldrich, CT, USA.
Tumor anti-initiating activity
Cytochrome P450 1A assay
CYP1A activity was assayed by the rate of dealkylation of 3-cyano-7-ethoxycoumarin (CEC) to the fluorescent
3-cyano-7-hydroxycoumarin according to [Crespi et al., 1997] and modified by [Gerhäuser et al., 2003].
Homogenates from cultured Hepa1c1c7 cells induced with β-naphthoflavone were used as a source of CYP1A.
Different S asperifolium polysaccharide extracts (10 μg/ ml as a final concentration) were incubated with the
Hepa1c1c7 cells homogenates. The rate of CEC conversion was measured kinetically by microplate
fluorescence reader (FLUOstar OPTIMA; BMG Labtech GmbH, Offenburg, Germany). Inhibition of CYP1A
activity was calculated in comparison with the initial fluorescence of a complete reaction mixture with cell
homogenate and buffer instead of the extract, and a-naphthoflavone was used as a reference inhibitor of
CYP1A activity.
Glutathione-S-transferases and glutathione evaluation
GST activity was measured according to the method previously described with [Habig et al., 1994] based on
GSTs catalyzed reaction between GSH and 1-chloro-2,4-dinitrobenzene that acts as an electrophilic substrate
for GSTs. GSH concentration was determined by the glutathione disulfide reductase/5,5'-dithiobis(2nitrobenzoic acid) enzymatic cycling procedure [Griffith, 1981]. HepG2 cells (1x106) were incubated with
different S. asperifolium polysaccharide extracts (10 μg/ ml as a final concentration) for 24 h. In the kinetic
analysis, the absorbance was assessed at 340 nm using microplate reader. GST and GSH were normalized to
the protein content as measured by bicinchoninic (BCA) acid assay [Smith et al., 1985].
Quinone reductase activity assay
After treatment Hepa1c1c7 cells with different S asperifolium polysaccharide extracts (10 μg/ml as a final
concentration) for 48 h, Hepa1c1c7 cells were washed by ice-cold phosphate-buffered saline (PBS) and
collected in buffer containing 25 mM Tris–HCl (pH 7.4) and 125 mM sucrose. Cells was sonicated for 15 s and
left in ice for 10 min. The homogenate was centrifuged for 20min at 13,000 ×g at 4 oC. QR activity was
determined in the supernatants by measuring the reduction of 2,6-dichloroindophenol [Yu et al., 2000]. Five
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microgram of protein, as determined by BCA, was added to 250 μl of the buffer (25 mM Tris–HCl, pH 7.4,
0.01% Tween 20, 60 μg of bovine serum albumin, 5 μM FAD, 80 μM 2,6-dichloroindophenol, and 0.2 mM
NADH). The reaction was performed at 25oC for 5 min and stopped by 30 μM dicumarol. Absorbance at 600
nm was measured with microplate reader. The specific QR activity was expressed as nmole of 2, 6dichloroindophenol reduced by 1mg of protein within 1 min.
Tumor anti-promoting and anti-inflammatory activity
Separation of macrophages from spleen
Single cell suspension was prepared from spleens of normal male C57BL/6 mice (pool of 3 mice). The
adherent monocytes were removed by two cycles of adherence of spleen macrophages to plastic petri dishes
(100 mm, Griener). For the 1st cycle, spleen macrophages were suspended in 15 ml complete RPMI-1640 and
dispensed as 5ml/ petri dish, then incubated for 2 h at 37°C in humidified air containing 5% CO 2 incubator. The
non adherent cells were collected; the plates were gently washed with warm media and subjected to another
round of incubation. The adherent cells were removed by scraping and centrifuged for 10 min at 2000 ×g. The
cells were re-suspended in incomplete phenol-red free RPMI-1640 and the viability was tested by trypan blue
stain (>85% viability).
Nitrite assay
Nitric oxide (NO) is an essential key mediator in the inflammation process. The accumulation of nitrites, an
indicator of NO synthesis, was measured by Griess reagent in macrophages that were grown in phenol red
free-RPMI media containing 10% FBS, according to the method of [Miranda et al., 2001]. Cells were incubated
for 24 h with bacterial lipopolysaccharide (LPS, 1 μg/ml) with or without different S asperifolium polysaccharide
extracts (10 μg/ml, as a final concentration). 50 μl of cell culture medium were mixed with 50 μl of Griess
reagent and incubated for 10 min. The absorbance was measured at 550 nm. A standard curve was blotted
using serial concentrations of sodium nitrite. The nitrite content was normalized to the cellular protein content.
The data was expressed as nmole/mg protein.
Determination of tumor necrosis factor-α
TNF-α was determined using sandwich ELISA as described by Markham et al., 1995, with some modifications.
The supernatants of LPS-stimulated macrophages treated with or without algal extracts (10 μg/ ml as a final
concentration), were collected after 20 h for TNF-α determination. A primary monoclonal antibody directed
against the N-terminal region of TNF-α (4 µg/ml) (Abcam, USA), was bound to a flat bottom 96- plate (50
µl/well) overnight at 4◦C. The plates were washed with washing buffer of 0.05% polyoxyethylene-20 (Tween-20)
in PBS and then blocking buffer (0.05% Tween-20, 5%fetal bovine serum in PBS) was added (200 µl/well) and
plates were incubated at 37◦C for 1.5 h. After washing, cell supernatants, recombinant TNF-α (0.195-100
ng/ml) were added (50 µl/well) and incubated at 37◦C for 2 h. After washing, the diluted secondary goat
antibody of high specificity for the C-terminus of antigen [biotin-labeled anti-TNF-α (200 ng/ml) polyclonal
antibody] was added as (50 µl/well) for 1 h incubation at 37°C. After washing, enzyme conjugate Streptavidinhorseradish peroxidase diluted 1:1000 (50 µl/well) (HRP, Koma Biotech Inc.) was added. After 1 h at 37°C, the
plates were washed, and the substrate solution was applied [equal volumes of 3, 3', 5, 5'-tetramethyl benzidine
(TMB) and H2O2; Kirkegaard and Perry Labs, Gaithersburg, MD] and added (50 µl/well). Color development
was stopped by 1 M HCl (50 µl/well) and measured at 450 nm using a microplate reader. TNF- α concentration
was proportional to the color developed in the standard curve recombinant TNF-α and then normalized to the
protein concentration in each sample. The data was expressed as ng/mg protein.
Tumor anti-progression activity
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Cytotoxicity assay
Cytotoxicity against HepG2 cell line was estimated by MTT assay, which react with mitochondrial
dehydrogenases in viable cells [Hansen et al., 1989]. HepG2 cells (5 x 104 cells/well) were incubated with
different S. asperifolium extracts (0–100 μg/ml, as a final concentration) at 37oC in a medium, before submitted
to MTT assay. The relative cell viability was expressed as the mean percentage of viable cells compared with
the control untreated cells. Then the half maximal inhibitory concentration (IC 50) was calculated from the
equation of the dose response curve.
Apoptosis assay
Annexin V is a protein that binds to phosphatidylserine (PS) residues, which are exposed on the apoptotic cell
surface, but not normal cells. In living cells, the PS groups in the plasma membrane are directed toward the
inside of the cell. During apoptosis, the PS groups are exposed to the exterior of the cell membrane. The
binding of PS with annexin V is therefore an established apoptosis marker, which was assayed by FITCconjugated Annexin V and PI double staining, using Apoptest Kit. PI is used to assess plasma membrane
integrity and cell viability, where it is excluded from cells with intact plasma membranes. HepG2 cells (5 x 10 4)
were collected after treatment with different S. asperifolium extracts (10 μg/ml, as a final concentration), for 6 h.
After treatment, cells were collected by trypsinization, washed with PBS, pooled with floating cells, submitted to
the kit procedure as manufacturer instructions, and then submitted to fluorescence microscope connected to
image analyzer.
Estimation of Histone deacetylase activity
The activity of histone deacetylase (HDAC) was measured in HepG2 cells using a colorimetric assay kit
(BioVision, Mountain View). The procedure involved the HDAC colorimetric substrate (Boc-Lys(Ac)-pNA), which
comprises an acetylated lysine side chain and was incubated with a sample containing nuclear extract.
Deactivation sensitized the substrate, and treatment with the lysine developer produced a chromophore, which
can be analyzed using microplate reader. HeLa cell nuclear extract was used as a positive control. A standard
curve was prepared using the known amount of the deacetylated standard (Boc-Lys-pNA). A similar volume of
control sample was added to 100 ng/ml trichostatin A (TSA), as a known inhibitor of HDAC.
Statistical analysis
Data analysis was conducted using (GraphPad Instat, version 3). For multiple comparisons, one way analysis
of variance (ANOVA) followed by Tukey-Kramer multiple comparison test was used. The results are expressed
as (Mean + SEM) and differences were considered significant at p value less than 0.05.
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Figures:
Fig. S1: The extraction steps of Sargassum asperifolium polysaccharides.
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