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Antioxidant and Anti-skin cancer potential
of a Ketocarotenoid pigment Astaxanthin
isolated from a green microalga
Haematococcus pluvialis Flotow
Infant Santhose, B.1, Elumalai, S.*2, Rajesh Kanna, G.3
1Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattangulathur,
Chennai – 603 203.
2Department of Biotechnology, University of Madras, Maraimalai Campus, Chennai - 600 039.
3Department of Plant Biology and Biotechnology, Presidency College (Autonomous), Chennai - 600
Abstract: Universally, astaxanthin is known for its powerful antioxidant potential which occurs naturally in various
marine and fresh water organisms. Haematococcus pluvialis, a unicellular green microalga is one of the efficient
producers of astaxanthin but comparatively in high concentration. The skin cancer is a major crisis coupled with direct
exposure of radiations from the sunlight due to ozone depletion. Especially, in Australia two/third of the people has
been diagnosed skin cancer below the age of 70. In this present study, the isolated pigment astaxanthin has been
appraised for the antioxidant and anti-skin cancer potential. About 90% of astaxanthin was extracted from the enriched
biomass of green alga Haematococcus pluvialis. The IC50 value of astaxanthin towards antioxidant and anticancer
activity was found to be 39.1 ± 1.14 µg/ml and 63 ± 0.22 µg/ml respectively. The gene expression fold of caspase 3
was enhanced further for different time periods of 0, 6 and 12 hours during the treatment of astaxanthin with the skin
cancer cells. While investigating the gene expression of oncogene Bcl-2 and tumor suppressor gene p53, the IC50
concentration of astaxanthin was found to suppress and induce both the genes respectively. Hence, it has been
proven that astaxanthin can prevent the proliferation of skin cancer cells.
Key Words: Haematococcus pluvialis; Astaxanthin; Skin Cancer; DPPH; MTT
Dr. S. Elumalai,
Professor and Head,
Department of Biotechnology,
University of Madras, Chennai
E mail ID: email@example.com
The incidence and dispersion of melanoma and non-melanoma skin cancer (MSC and
NMSC) is the major public health concern among people (Young et al., 2005). About 90% of the
MSC and NMSC are caused by the carcinogenesis due to enormous UV exposure (Skin cancer
foundation, 2008). The rate of NMSC was increased by 3-8% annually in Europe, US, Canada
and Australia since 1960 (Diepgen and Mahler, 2002). Several epidemiological studies have
linked the rising rates of NMSC with ozone depletion, increased exposure to UV light, changes
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in clothing style, increased longevity, and increased outdoor activities (Diepgen and Mahler,
Astaxanthin is a symmetric ketocarotenoid, (3, 3’-dihydroxy-β, β’-carotene- 4,4’-dione)
found predominantly in marine forms including microalgae, trout, salmon, shrimp, krill, crayfish
and crustaceans (Breithaupt, 2007). Natural astaxanthin is usually found either conjugated to
proteins or esterified with fatty acids. Since, the consumers of aquatic ecosystem are not capable
of carotenoid de novo synthesis where the xanthophyll and other carotenoid pigment found in
their bodies (e.g. canthaxanthin and lutein). Hence, microorganisms (producers) being the main
source of carotenoid for them (Johnson and An, 1991). The carotenoid pigment isolated from the
microalga Haematococcus sp. was named as “haematochrom” till 1944 and which was identified
as astaxanthin a principle carotenoid pigment accumulated quantitatively high in the microalga
Haematococcus sp. up to 4-5% cell dry weight (Tisher, 1944). In 1954, Droop described the
conditions governing astaxanthin formation. He showed that the action of light and carbon
dioxide were dependent on one another, but that of organic carbon (such as acetate) is
independent of light.
Thus, astaxanthin formation could occur in the dark when energy is derived from organic
carbon. Encystment and astaxanthin production can be induced by low nitrate or phosphate, high
temperature or light, or the addition of sodium chloride in the culture medium (Boussiba and
Vonshak, 1991; Kobayashi et al., 1992; Fan et al., 1994; Kakizono et al., 1992). Studies have
demonstrated that the significant role played by natural carotenoid in regulating immunity and
disease etiology. Astaxanthin significantly reduces DNA damage, stimulates lymphocyte
proliferation and increases natural killer cell cytotoxic activity produces increased number of T-
cells and enhances the total number of antibody producing B-cells.
In addition, it also has pro-vitamin-A property more effective antioxidant than other carotenoids,
decelerate age-related macular degeneration, immunomodulatory effects, etc. (Lorenz and
Cysewsky, 2000; Dufosse et al., 2005; Breithaupt, 2007). The salivary immunoglobulin A (sIgA)
is an antibody plays a major role in the immunity; where the supplementation of astaxanthin to
the soccer players have shown elevated levels of sIgA and hampers muscle damages by their
anti-inflammatory potential (Baralic et al., 2015).
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Therefore, the astaxanthin is used in pharmaceutics, neutraceutics, cosmetics and food
additives with a recent great success on the market (Dufosse et al., 2005). During March 2010,
researchers at Washington State University showed that astaxanthin inhibits cancer cell growth
by decreasing free radical induced cellular damage, reducing inflammation and increasing
immune response. Various drugs are being implied to treat skin cancer, but astaxanthin is a
wonder drug due to its strong free radical quenching property. A431 skin cancer cell line is a
model cell line (epidermoid carcinoma) used in biomedical research. They contain no
functional p53, a potent tumor suppressor gene, and so are highly sensitive to mutagenic stimuli.
A431 skin cancer cells were established from an epidermoid carcinoma in the skin/epidermis of
an 85- year-old female patient ("A431 - Cytokines and Cells Online Pathfinder Encyclopedia").
In this present study, the isolated ketocarotenoid pigment astaxanthin from the green microalga
Haematococcus pluvialis was evaluated for its antioxidant and anticancer activity against a skin
cancer cell line A431.
2. Materials and Methods
2.1: DPPH free radical scavenging assay
The free radical scavenging potential of astaxanthin (test sample) was determined by the
DPPH free radical scavenging assay (Blois, 1958). The DPPH free radical scavenging activity of
the pigment astaxanthin was calculated in terms of percentage of inhibition of the free radicals
by using the following formula.
Percentage of inhibition (%) = (Absorbance of Control - Absorbance of Sample) × 100
Absorbance of Control
2.2: Apoptotic cell-toxicity assay
The cell viability of the skin cancer cell line A431 was assessed against the test sample
astaxanthin based on the MTT assay described by Mosmann in 1983. The absorbance of purple
blue formazan dye was measured by Microplate reader at 570 nm (Biorad 680). The cytotoxicity
was determined using Graph pad prism5 software. The 50% inhibitory concentration value (IC50)
of the test sample (astaxanthin) was evaluated and recorded.
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2.3: DNA laddering assay
The A431 skin cancer cells were treated with different concentrations of astaxanthin (25
µl to 250 µl). Then the cells were nourished with DMEM medium and incubated for 24 hours.
The genomic DNA was extracted and separated with 2 µl of DNA ladder electrophoretically
using 1.2 % of agarose gel at 100 V. The separated DNA bands were observed at 312 nm in a
2.4: Caspase activity assay
The activity of caspase-3 belongs to A431 skin cancer cells were determined
chromogenically by cleavage of substrate: Ac-DEVD-7-amino-4-methylcoumarin pNA. (Short
caspase specific peptides) according to described methods (Kohler et al., 2002). Cleavage of the
chromogenic peptide substrates was monitored by pNA release in a 405 nm scan II plate reader
(Biorad, USA). Chromogenic units were converted to percentage by comparing the untreated
control cells generated free pNA. Quantification of caspase activity was calculated as fold
increase over control samples.
2.5: Gene expression assay
Total RNA was isolated using TRIZOL-(Sigma, India) according to the manufacturer’s
instructions. Frequently, the test sample (astaxanthin) in TRIZOL was repeatedly pipetted in and
out to disrupt the A431 skin cancer cells. The test samples (astaxanthin) were incubated for 5
min. at room temperature to permit complete dissociation of nucleoprotein complexes. RT-PCR
was performed in triplicate using SuperScriptTM two Step RT-PCR with platinum® Taq kit
according to the manufacturer’s recommendations (Invitrogen, Carlsbad, CA, U.S.A.).
For cDNA synthesis, Complementary DNA was synthesized from 1 µg total RNA from
each sample in 20 µL of reaction buffer (contained 50 mM Tris-HCl, pH 8.3, 75 mM KCl and 3
mM MgCl2) using SuperScript II reverse transcriptase enzyme (Genetech, RT-PCR mix-
Germany) in a 20 µl volume reaction containing 10 mM dithiothreitol, 10 U RNase inhibitor
(Promega, Madison, WI, USA), 1 mM dNTPs and 2.5 µM random hexamers. The cDNA (1 µl)
was then amplified in 20 µl of reaction buffer for 35 cycles of denaturation (94°C for 30 s),
annealing (50°C for 30 s), and extension (72°C for 30 s) using the following primers: Primers
used were b-actin (FW 5’ -ATGTTTGAGACCTTCAACAC- 3/ RW 5’ -
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CACGTCACACTTCATGATGG- 3’) (expected product 489 bp). p53: FW, 5'-
AGGGTTAGTTTACAATCAGC-3', RW, 5'-GGTAGGTGCAAATGCC-3'; bcl-2 FW, 5'-
TCGATGTGATGCCTCTGCGAA GAAC-3'; RW, 5'- ATTGCACTGCCAAACGGAGCTG-3';
The cDNA amplified as described above were run on a 1.2% agarose/EtBr gel in 1X TAE
(Tris-acetate-EDTA) buffer and then visualized under UV illuminator (Sambrook and Russel
2001). The density of each band on the agarose gel was measured using ImageJ pixel
quantitation software. Background measurements were subtracted, and a relative number was
assigned to each band intensity (Grifantini et al., 2003).
3.1. DPPH free radical scavenging activity
The 50 % inhibitory concentration (IC50) of the astaxanthin required to hamper the
generation of free radicals was found to be 39.1 ± 1.14 µg / ml (Fig. 1) when compared to a
positive control (ascorbic acid). The free radical scavenging potential of the astaxanthin isolated
from H. pluvialis was found auspicious to control the emerging free radicals.
DPPH chelating activity
Test Samples in µg/ml
DPPH in Percentage (%)
Fig. 1: DPPH free radical scavenging activity of astaxanthin in comparison with a positive
3.2. Apoptotic cell-toxic activity
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The apoptotic toxicity of the skin cancer cells remained significant with 22.080±4.760 %
and 33.52 ± 2.32 % upon astaxanthin treatment with the concentration of 500 µg/ml and 100
µg/ml on A431 skin cancer cells respectively (Fig. 2). The inhibitory concentration IC50 and IC30
values to prevent the proliferation of skin cancer cells were found to be 63 µg/ml and 37.8 µg/ml
Concentration in µg/ml
% of cell viability
Fig.2: Apoptotic cell-toxicity assay of astaxanthin on A431 skin cancer cell lines
3.3. DNA Laddering Assay
The experiment was characterized by the activation of endogenous endonucleases with
subsequent cleavage of the chromatin DNA into inter-nucleosomal fragments of roughly 50 base
pairs (bp) and multiples thereof (100, 150 etc.). This method of DNA laddering assay can be
used to detect the apoptosis of cancer cells due to the treatment of the test compound. The gel
image (Fig. 3) particularly lane 3 and 4 shows the fragmented and drifted DNA due to the
apoptotic activity of cancer cells by the treatment of the astaxanthin extracted from the green
microalga Haematococcus pluvialis.
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Fig. 3: The agarose gel image showing bands of DNA, lane 1: DNA ladder; lane 2: normal
A431 skin cancer cell’s DNA; lane 3 and 4: A431 skin cancer cell’s DNA treated with the
test compound astaxanthin
3.4. Caspase activity assay
The mRNA levels of caspase 3 were evaluated for better understanding the action of
astaxanthin over apoptosis of skin cancer cells. The mRNA expression fold of caspase 3 was
compared with the control cells (untreated cells) and analyzed for 0, 6 and 12 hours and the
values were 1.05±0.05, 1.37±0.03 and 1.795±0.075 respectively (Fig. 4). Hence, the result
confirmed that the caspase 3 expression was elevated while increasing the time of incubation
with the test compound astaxanthin.
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Time in hours
Fig. 4: Caspase activity assay showing the expression fold of caspase 3 gene
3.5. Gene expression assay
A group of genes P-53, Bcl-2, NFkB and Beta actin were chosen for the gene expression
studies. The gene expression was compared with the house keeping gene beta actin as an internal
control (Fig. 5). The gene expression of p53, Bcl-2 and NFkB were compared between the
astaxanthin treated and the untreated cells of A431 skin cancer cells. The activity of the tumor
suppressor gene p53 was increased to two folds while Bcl-2 is an oncogene whose activity is
reduced and NFkB is a gene which is responsible for cell survival and there is no such
considerable change in the astaxanthin treated A431 skin cancer cells (Fig. 6). Therefore, the test
compound astaxanthin is determined to be effective against the skin cancer cell line A431.
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Fig. 5: The agarose gel image with bands showing the expression fold of different genes,
lane 1: DNA ladder; lane 2: Control (normal cDNA genes) lane 3: bands from A431 skin
cancer cells and lane 4: bands belongs to A431 skin cancer cells treated with astaxanthin
Fig. 6: The IC30 and IC50 values of astaxanthin treated A431 skin cancer cells showing
expression fold of three genes p53, Bcl-2 and NFkB, where the suppression of Bcl-2 and
over-expression of p53 was obtained at IC50
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Due to strong free radical scavenging property of astaxanthin, it has been proved as a
neutraceutical and pharmaceutical agent against free radical supported diseases including
cardiovascular diseases, neuro-degenerative diseases, oral, colon and liver cancers (Lorenz and
Cysewski, 2000; Guerin et al., 2003). During such high oxidative stress conditions, the
astaxanthin and its esters has proven to be a strong free radical scavengers which can restore the
inactivated antioxidant enzymes. The ROS quenching enzymes such as superoxide dismutase,
catalase, peroxidase and thiobarbituric acid reactive substances (TBARS) were obtained in high
concentration in the blood plasma of rat while feeding with biomass of H. pluvialis as a source of
astaxanthin (Ranga Rao, 2010). The astaxanthin proved against the hydroperoxides and
superoxide free radicals and which is even high in diabetic patients (Hashimoto et al., 2013). The
age related macular degeneration was treated with astaxanthin and resulted that the cells were
well protected against the oxidative stresses (Li et al., 2014).
The oral supplementation of astaxanthin protects the retinal photoreceptors of eyes when
exposed to UV (Tso and Lam, 1996); prevents UV-induced photooxidation and protect skin and
eggs of salmon fish (Connor and Brien, 1998). In this present study, the DPPH free radical
scavenging assay has significantly proven the antioxidant potential of the test compound
astaxanthin isolated from green microalga Haematococcus pluvialis. The antioxidant results
from the present investigation paved the way to analyze the cell viability of the skin cancer cells
using the MTT assay using a skin cancer cell line A431. Hence, the cell viability of the skin
cancer cells was found to be decreased by increasing the concentration of the astaxanthin which
significantly reveals the enhancement of apoptosis of skin cancer cells.
The Caspase-3 is commonly activated by numerous death signals and cleaves a variety of
important cellular proteins among the caspases identified. Failure of caspase mediated apoptosis
is one of the main contributions to tumor development and autoimmune diseases (Kumar, 2007
and Wang et al., 2005). The agarose gel image from this present study has shown the
fragmentation of DNA by DNA Laddering assay which indicates the activity of the caspase
activated DNase (CAD) during astaxanthin treatment with the skin cancer cells. Therefore, the
mRNA expression fold of caspase 3 was found to be enhanced by the treatment of astaxanthin
and further increased by increasing the duration of the treatment. As a result from this present
study, the apoptotic cells may form a ladder of DNA fragments during electrophoretic
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separation. Thus, the astaxanthin has proven to be a potent antitumor drug to cause DNA damage
to the cancer cells (Jamieson and Lippard, 1999).
The different types of carotenoid pigments have already been reported to hamper the
propagation of human breast cancer MCF-7 cell line in vitro (Li et al., 2002) especially,
astaxanthin was reported to minimize the occurrence of pre-neoplastic lesions and neoplasm in
mice with bladder cancer. The benign prostate cancer was caused due to the over expression of
5-α-reductase enzyme which was suppressed by the supplementation of astaxanthin (Anderson et
al., 2001). Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and
interfering with programmed cell death independent of promoting cell division (Hockenbery et
al., 1990). Bcl-2 inhibits most types of apoptotic cell death, implying a common mechanism of
lethality. Bcl-2 is localized to intracellular sites of oxygen free radical generation including
mitochondria, endoplasmic reticulum and nuclear membranes (Hockenbery et al., 1993).
Whereas, NFkB (nuclear factor kappa beta) is a transcription factor that plays important roles in
the immune system (Bonizzi and Karin, 2004) and a ubiquitous transcription factor involved in
proliferative signaling and tumor promotion and is activated by oxidants and other stimuli known
to generate ROS (Dhar et al., 2002) Moreover, pathological dysregulation of NFkB is linked to
inflammatory and autoimmune diseases as well as cancer.
This present investigation upon the evaluation of astaxanthin towards the apoptosis of
skin cancer cells was focused on the expression of such genes including p53, Bcl-2 and NFkB.
The astaxanthin upon the treatment of skin cancer cells has enhanced the expression of the p53
gene responsible for the suppression of tumor induction. At the same time the expression of an
oncogene Bcl-2 was hampered and there is no significant modulation in the expression of gene
NFkB responsible for autoimmune system. Furthermore, animal model studies are much needed
to evaluate the potential activity of astaxanthin on skin cancer treatment.
Therefore, the overall investigation infers that the ketocarotenoid compound astaxanthin
isolated from a green microalga Haematococcus pluvialis is very potent to suppress the
proliferation of the skin cancer cells and thereby to enhance the apoptosis of skin cancer cells.
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6. Conflict of interest
According to the authors there is no conflict of interest.
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