Biomedicine & Preventive Nutrition 2 (2012) 149153

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Original article

Antiproliferative potential of astaxanthin-rich alga Haematococcus pluvialis

Flotow on human hepatic cancer (HepG2) cell line
S. Nagaraj a, , M.G. Rajaram a , P. Arulmurugan a , A. Baskaraboopathy a , K. Karuppasamy b ,
K.R. Jayappriyan a , R. Sundararaj c , R. Rengasamy a
a
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, India
b
School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai, India
c
Government Arts & Science College, Nandanam, Chennai, India

a r t i c l e i n f o a b s t r a c t

Article history: To investigate the efcacy of astaxanthin from unicellular green alga, Haematococcus pluvialis inhibited
Received 25 February 2012 the cell growth and trigger the apoptosis in human hepatoma cancer cell line (HepG2) in a dose and time
Accepted 30 March 2012 dependent manner. Cell viability was analyzed by 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyl-tetrazolium
bromide (MTT) assay. The different concentration of astaxanthin viz. 5, 10, 15, 20 and 25 g/mL to incubate
Keywords: the cell lines at different time course. The lactate dehydrogenase leakage (LDH) content and reduced
Haematococcus pluvialis glutathione (GSH) levels were also estimated. The depletion of GSH may be involved in the induction
Astaxanthin
of apoptosis of the cancer cells. The apoptosis was evaluated with Annexin-V and Propidium iodide
Liver cancer
Annexin-V
under uorescent microscopy study. Moreover, the efcacy of astaxanthin 25 g/mL signicantly inhibits
Apoptosis the hepatoma cancer cell growth and induces cell apoptosis. In addition, immunouorescence staining
and ow cytometry studies were revealed that further evidence to conrm that the astaxanthin 15 and
25 g/mL to trigger the apoptosis 42.55% and 58.55% for 24 h, respectively. Taken together, the present
study suggests that H. pluvialis astaxanthin may protect from liver cancer.
2012 Elsevier Masson SAS. All rights reserved.

1. Introduction UV light photo-oxidation, inammation, cancer, aging and age-

The micro green alga Haematococcus pluvialis Flotow fresh
water, motile unicellular has a commercial importance owing
to its ability to accumulate ketocarotenoids, astaxanthin, which
is biologically active [1]. Astaxanthin is closely related to other
well-known carotenoids, such as - carotene, zeaxanthin and
lutein, thus they share many of the metabolic and physiological
functions attributed to carotenoids. Astaxanthin are extensively
used in various applications in nutraceutical and pharmaceuti-
cal industries due to its superior antioxidant activity [2,3]. The
compound is widely employed as a pigmentation inducer, and
due to its good biological function such as antioxidant acti-
vity, it has been used for immune response enhancement and
cancer protection [4]. Astaxanthin was shown to have 500-fold
stronger free radical antioxidant activity than vitamin E and
38-fold than -carotene [5]. The antioxidant properties of astax-
anthin are believed to have a key role in protection against
Corresponding author. Centre for Advanced Studies in Botany, University of
Madras, Guindy campus, Lab # 402 Chennai, 600 025, India.
E-mail addresses: nagalilly@gmail.com, nagus80@yahoo.com (S. Nagaraj).
related macular degeneration, or enhancement of the immune
response, liver function, and heart, eye, joint, and prostate health
[3].
The methods reported in the literature for the determination
of astaxanthin from encysted secondary carotenoid accumulation
phase can be used to quantify pigments extracted by mechanical
and solvent methods [6]. The extracted pigments are then subjected
further separation and identication by thin layer chromatography
(TLC), UVvisible spectrophotometry and high-performance liquid
chromatography (HPLC). The measurement of pigment astaxanthin
in H. pluvialis cells has already been reported [79]. In fact, scientic
reports indicate that astaxanthin, because of its antioxidative pro-
perties, has antitumor [10] and anti-inammatory [11] activities,
positive effects on blood pressure [12] as well as cardioprodective
effect [13]. Accordingly, in this ndings, we investigate the effects
of extracted astaxanthin from H. pluvialis and the application of
antitumor activity of human cancer cell lines of hepatocellular car-
cinoma (HepG2), breast cancer cell lines of (MCF-7) and antioxidant
activities, in vitro, of the astaxanthin were evaluated by MTT (3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay
and DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging
assay.

2210-5239/$ see front matter 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.bionut.2012.03.009
150 S. Nagaraj et al. / Biomedicine & Preventive Nutrition 2 (2012) 149153

2. Materials and methods

2.1. Algal culture

H. pluvialis was obtained from the Culture Collection of Algae,

Centre for Advanced Studies in Botany, University of Madras, Chen-
nai. The culture was maintained in Bold Basal Medium (BBM) pH
7.5, temp., 2325 C and 18/6 h light and dark conditions. Extraction
of carotenoids method was followed by [14].

2.2. Chemicals and reagents

Dimethy sulfoxide (DMSO), MTT, Annexin-V and Propidium

iodide were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). DMEM medium, fetal bovine serum, penicillin and strepto-
mycin were purchased from Invitrogen (Carsbad, CA, USA). All other
reagents were of analytical grade.

2.3. Estimation of carotenoids

The absorbance of the extracts was read at 476 nm using a Milton

Roy UV-Spectrophotometer. Total carotenoid contents were calcu-
lated by method of [15]. Astaxanthin was analyzed by the method
of [14]. Extractability of astaxanthin was calculated for all samples
as per the procedure of [16] by the formula.
Fig. 1. Thin layer chromatography prole of Haematococcus pluvialis astaxanthin.

2.4. Separation of carotenoids by thin layer chromatography

(TLC) 2.8. Lactate dehydrogenase leakage assay

The H. pluvialis extract were analysed by using TLC aluminium
sheets (10 10 cm) precoated with silica gel 60 (Merck Ltd, New
Delhi). H. pluvialis extract was spotted on TLC sheet and deve-
loped using solvent system acetone: n hexane (3:7 [17]). The elute
allowed to dry at room temperature and carotenoids were identi-
ed by comparing with plates were identied by comparing with
authentic astaxanthin (Fig. 1).
2.5. Drug preparation
Lactate dehydrogenase (LDH) leakage assay was performed by
the method of [19]. One hundred microliters of conditioned media
of control and astaxanthin treated HepG2 cells were added to 1-
mL cuvette containing 0.9 mL of a reaction mixture to yield a nal
concentration of 1mmol/L pyruvate, 0.15 mmol/L NADH and 104
mmol/L phosphate buffers, pH 7.4. After thoroughly mixed, the
absorbance of the solution was measured at 340 nm for 45 s. LDH
activity was expressed as micromoles of NADH used per minute
per well.

Acetone extract of astaxanthin was dissolved in 1% DMSO (nal
concentration of the DMSO did not exceed 1% [v/v] and did not
affect the cell proliferation) prepared in serum-free DMEM medium
and ltered by 0.3 mm syringe lter and stored.
2.6. Cell lines and culture maintenance
Human hepatoma cell lines (HepG2) were procured from
National Center for Cell Science, Department of Biotechnology,
Pune, India. Cells were grown as monolayers in DMEN medium,
supplemented with 10% (v/v) heat inactivated FBS, antibiotics
(penicillin 100 U/mL, streptomycin 10 g/mL) and 1 mmol/L
sodium pyruvate under standard conditions (37 C) in a controlled
humidied atmosphere containing 50 mL/L CO2 .
2.7. Growth inhibition assay
The inhibition effects of astaxanthin on the growths of HepG2
cells cytotoxicity of the extraction of astaxanthin were assessed
by cell viability test using MTT assay [18]. For the determina-
tion of cell viability, cells were plated at the density of 5 104
cells/well and cultured for 24 and 48 h. The medium was replaced
with serum-free medium (DMEM medium, supplemented with
antibiotics [penicillin 100 U/mL, streptomycin10 g/mL, 1 mmol/L
sodium pyruvate]) and the cells were treated with various concen-
trations of astaxanthin (5, 10, 15, 20 and 25 g/mL) for 24 h and
48 h and incubated with 1% DMSO as solvent control.
2.9. Estimation of reduced glutathione
Total reduced glutathione (GSH) was determined by the method
of [20]. Five percent TCA was added to HepG2 cells (1 104 cells).
The precipitate was removed by centrifugation. To an aliquot of
the supernatant, 2 mL of DTNB reagent was added to make a nal
volume of 3 mL. The absorbance was read at 412 nm against a blank
containing TCA instead of sample. Aliquots of the standard solu-
tion were treated similarly. The amount of GSH was expressed as
nmoles/104 cells.
2.10. Fluorescent microscope analysis
Apoptotic morphology was studied by staining the cells with
a combination of the uorescent DNA-binding dye. After treat-
ment with astaxanthin (15 and 25 g/mL for 24 h), cells were
collected, washed and suspended in phosphate buffer saline (PBS).
After stained with the equal mixture of Annaxin-V and Propidium
iodide (each dye was dissolved in 100 g/mL of PBS), the cells were
examined [21]. The differential uptake of these two dyes allowed
the identication of viable and non-viable cells under uorescent
microscope and the results were recorded.
2.10.1. Flow cytometry analysis (FACS)
Flow cytrometry analysis was done as described by [22]. Ato-
tal of 1.0 106 HepG2 cells were grown in a 6-well plate and 15
and 25 g/mL of astaxanthin obtained from H. pluvialis were added.
 S. Nagaraj et al. / Biomedicine & Preventive Nutrition 2 (2012) 149153
Plate was incubated for 24 h. The cells were then lysed with 0.1%
TritonX-100 and incubated with 50 g/mL RNase A for 30 min at
37 C. Nuclear DNA was stained with 50 g/mL Propidium iodide
and the cells were analysed for DNA content using uorescence
activated cell sorter (FACS Calibur System, BD Biosciences) and
analyzed using Cell Quest Software (BD Biosciences).
2.10.2. DNA fragmentation
DNA fragmentation was followed by the method of [23]. The
HepG2 cells were placed in a 60-mm culture dish at a density of
5 106 cells and treated with astaxanthin (15 and 25 g/mL) for
24 h. The cells attached at the bottom were scraped off and collected
together with unattached cells by centrifuging at 1500 rpm/min for
5 min at 4 C. DNA was prepared from the pelleted cells. Briey, the
cells were lysed with lysis buffer and extracted with 2 mL of phe-
nol (neutralized with TE buffer, pH 7.5) followed by extraction with
1 mL of chloroform and isoamyl alcohol in the ratio of 24:1. The
aqueous supernatant was precipitated with 2.5 volume of ice-cold
ethanol with 10% volume of sodium acetate at 20 C overnight.
After centrifugation at 13,000 rpm/min for 10 min, the pellets were
air-dried and resuspended with 50 L of TrisEDTA (TE) buffer.
Equal quantities of DNA were electrophoresed in 1.8% agarose gel
containing 0.5 mg/L of ethidium bromide. After electrophoresis, the
gel was photographed under UV-light carefully.
2.10.3. Statistical analysis
Values were expressed as mean SD. Difference between the
drug-treated group and control group was analyzed using [24] soft-
ware. P < 0.05 was considered statistically signicant.
3. Results
3.1. Growth inhibition on the HepG2
The cytotoxicity of astaxanthin on HepG2 cells were examined
by MTT assay method. Fig. 2 shows the viability of control and astax-
anthin treated (5, 10, 15, 20 and 25 g/mL) HepG2 cells. The alga
extract induced cytotoxicity to the human liver cancer HepG2 cells
in a concentration-dependent manner after 24 and 48 h of treat-
ment. The results showed that treatment with astaxanthin could
increase the number of apoptotic cells and decrease the number of
normal cells. Apoptotic cells were detached from the surface and
suspended in the medium.
3.2. Lactate dehydrogenase leakage assay
The levels of LDH released into the medium of control and
astaxanthin treated (5, 10, 15, 20 and 25 g/mL) HepG2 cells are
presented on Fig. 3. LDH activities were signicantly elevated after
Fig. 2. Cell viability analysis of HepG2 human liver cancer cell line.
151
Fig. 3. Lactate dehydrogenase leakage (LDH) level in HepG2 cells were exposed to
different concentrations of astaxanthin (from 5 g/mL to 25 g/mL) from Haemato-
coccus pluvialis for 24 h. Results were expressed as the percent of total cell number
and the percentage of LDH leakage was analyzed. Values are mean SD (n = 6).
P < 0.05 represents the statistical signicance between control and astaxanthin
treated cells.
24 h of exposure to astaxanthin in the medium when compared to
the control.
3.3. Reduced glutathione (GSH) levels
The levels of GSH content in HepG2 cells control was
14.11 3.26 and 19.17 2.71 nmoles/104 cells, 24 h and 48 h
respectively. After treatment with astaxanthin (15 and 25 g/mL)
24 h and 48 h, the GSH levels were recorded in 21.15 2.54 and
28.23 4.37 nmoles/104 cells and 32.82 6.34 and 49.81 5.51
nmoles/104 cells, respectively. There was a signicant difference
between the astaxanthin treated group and control group (Table 1).
3.4. Fluorescence microscopic observation
The morphological changes were observed in HepG2 cells
treated with astaxanthin 15 and 25 g/mL for 24 h. As shown on
Fig. 4, normal live cells were bright green in color whereas astaxan-
thin treated cells were bright orange to red in color with condensed
nuclei. Besides, normal nuclei showed chromatin with an orga-
nized structure, while apoptotic nuclei showed highly condensed
chromatin in the treated cells.
3.5. Fluorescent Activated Cell Sorting (FACS) analysis
Apoptosis (cells with broken DNA) and cell cycle distribution in
HepG2 cells were studied after treated with H. pluvialis astaxanthin
for 24 h. For HepG2 cells, a signicant arrest at G0/G1 was observed
at 24 h of treatment, whilst the number of cells in S and G2-M
phases were reduced signicantly, but increased apoptosis (sub-
G1 phase) was not observed at this time. Treatment with 15 and
25 g/mL of astaxanthin for 24 h signicantly increased the propor-
tion of cells with a reduced DNA content (sub-G0/G1 or A0 peak),
indicative of apoptosis with loss of cells in the G1 phase. Loss of DNA
occurs as a result of DNA fragmentation from the cells after endonu-
clease cleavage. After staining with propidium iodide, these cells
would have taken up less stain and appear in sub-G0/G1. Incubation
Table 1
Intracellular reduced glutathione levels in HepG2 cells after 24 h and 48 h treatment
with astaxanthin from Haematococcus pluvialis.
Cell line (HepG2) Control 15 g/mL 25 g/mL
24 h 14.11 3.26 21.15 2.54 32.82 6.34
48 h 19.17 2.71 28.23 4.37 49.81 5.51
Values are given statistically signicant at P < 0.05 (n = 3).
152 S. Nagaraj et al. / Biomedicine & Preventive Nutrition 2 (2012) 149153
Fig. 4. Morphological observation of HepG2 cells under uorescence microscope. The cells treated with astaxanthin (15 g/mL and 25 g/mL) for 24 h. Treated cells were
stained with propidium iodide and Anexine-V.
of the cells with 15 to 25 g/mL of astaxanthin for 24 h signi-
cantly increased the proportion of cells with a reduced DNA content
from 24.27% (control) to 42.55% (15 g/mL astaxanthin) and 58.55%
(25 g/mL of astaxanthin) for 24 h treatment. In cells treated with
H. pluvialis, apoptosis was induced in a dose-dependent manner, as
evidenced by the method. The pro-apoptotic effects were observed
evidently at concentrations of H. pluvialis of 15 and 25 g/mL.
3.6. DNA fragmentation
In the control, no more fragmentation was observed in
agarose gel. However, the signal appearing in astaxanthin (15 and
25 g/mL) 24 h treated HepG2 cells showed the fragmented lad-
dering pattern of DNA, indicating the characteristics of apoptotic
cells (Fig. 5).
4. Discussion
In the present study, we have investigated the effect of astaxan-
thin on proliferation and apoptosis of HepG2 liver cancer cell line.
The cytotoxic effect of algal astaxanthin was tested by MTT assay.
A signicant decrease was noticed on the cell viability between 5
to 50 g/mL of algal astaxanthin. At 25 g/mL of algal astaxanthin
Fig. 5. DNA fragmentation in HepG2 cells treated with astaxanthin from Haemato-
coccus pluvialis. Lane A. Control HepG2 cells without any treatment. Lanes B and C.
15 g/mL and 25 g/mL 24 h respectively.
treatment, only 50% of cells were viable at the end of 48 h. The IC50
value of algal astaxanthin was calculated as 25 g/mL. Treatment of
the cells with astaxanthin interfered with the normal reorganiza-
tion of microtubule network, and inhibits the formation of normal
spindle at metaphase required for mitosis and cell proliferation.
These effects lead to the arrest of the cells in G2/M phase of the cell
cycle and eventually to apoptotic cell death [2529].
LDH, a cytoplasmic enzyme that catalyzes the oxidation of lac-
tate to pyruvate and vice versa, is a known marker of membrane
integrity and the regulator of vital biochemical reactions [30]. LDH
leakage monitors the integrity and permeability of the plasma
membrane and it is sensitive and easy to perform. In the present
attempt, the effect of astaxanthin was made on LDH of HepG2 con-
ditioned medium for 24 h. The activity of LDH was decreased in
cell lysate when compared to control cells from 5 to 50 g/mL of
algal astaxanthin treated cells. However, the activity of LDH was
increased in conditioned medium when compared to control cells.
This conrmed the cytotoxic effects of algal astaxanthin against the
HepG2 cells. It indicated that the algal astaxanthin increased the
plasma membrane permeability and therefore there was a leakage
of LDH from the cells into the medium in turn increased the activity
of LDH.
Recent studies have provided evidence that GSH plays a distinct
role in the resistance of certain tumors to chemotherapy. Deple-
tion of intracellular GSH can result from a number of mechanisms
including inhibition of cysteine uptake, inhibition of glutathione
synthesis, MRP-mediated efux, GSH oxidation, and transthiola-
tion reactions leading to glutathione-protein adducts [3133]. To
prove this hypothesis for the liver cancer cell line HepG2, the
dietary carotenoids astaxanthin from H. pluvialis treated with 15
and 25 g/mL for 24 h and 48 h. The maximum depletion of GSH
was noted in the concentration of 25 g/mL at 48 h recorded in
49.81 5.51 nmoles/104 cells. There was a signicant difference
between the astaxanthin treated group and control group. In addi-
tion to these, GSH depletion may also play a vital role in apoptosis
through redox imbalance in HepG2 cells.
Apoptotic cells often produce a unique ladder composed of
nucleotide fragments at an interval of 180200 base pairs, which
can be visualized by DNA-agarose gel electrophoresis. It is generally
assumed that the toxicity of antitumor drugs is the consequences
of their ability to cause genomic DNA damage in cancer cells
[34]. In this context, Bai and Cederbaum [35] reported that many
chemotherapeutic drugs, including Vp16 and mitomicin C, induce
cells to undergo apoptosis through damage of nuclear DNA. Panno
[23] have reported that low dose also induced the DNA damage
through apoptosis.
In the present study, HepG2 cells treated with 15 and 25 g/mL
of H. pluvialis astaxanthin for 24 h induced an extensive DNA frag-
mentation. However, the control cells showed intact DNA. Thus, it
was conrmed that the cell death was caused by algal astaxanthin
through apoptosis. A signicant arrest at G0/G1 was observed on
 S. Nagaraj et al. / Biomedicine & Preventive Nutrition 2 (2012) 149153
HepG2 cells at 24 h of treatment, whilst the number of cells in S
and G2-M phases were reduced signicantly, but increased apop-
tosis (sub-G1 phase) was not observed at this time. Treatment with
15 and 25 g/mL of astaxanthin for 24 h signicantly increased the
proportion of cells with a reduced DNA content (sub-G0/G1 or A0
peak), indicative of apoptosis with loss of cells in the G1 phase.
Loss of DNA occurred as a result of DNA fragmentation from the
cells after endonuclease cleavage. After staining with propidium
iodide, these cells were less stained and appeared in sub-G0/G1.
Incubation of the cells with 15 to 25 g/mL of astaxanthin for 24 h
signicantly increased the proportion of cells with a reduced DNA
content from 24.27% (control) to 42.55% (15 g/mL astaxanthin)
and 58.55% (25 g/mL of astaxanthin) at 24 h treatment. Thus pro-
apoptotic effects were conrmed at the concentrations of 15 and
25 g/mL of H. pluvialis astaxanthin in the present attempt. Thus,
the present investigation revealed that signicance of inclusion of
H. pluvialis astaxanthin in the formulation of drugs to treat liver
cancer.
Disclosure of interest
The authors declare that they have no conicts of interest con-
cerning this article.
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