Marine Microalgae with Anti-Cancer Properties
Kevin A. Martínez Andrade, Chiara Lauritano ID , Giovanna Romano and Adrianna Ianora *
Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy;
kevin.martinez@szn.it (K.A.M.A.); chiara.lauritano@szn.it (C.L.); romano@szn.it (G.R.)
* Correspondence: adrianna.ianora@szn.it; Tel.: +39-081-583-3246
Received: 23 April 2018; Accepted: 12 May 2018; Published: 15 May 2018
Abstract: Cancer is the leading cause of death globally and finding new therapeutic agents
for cancer treatment remains a major challenge in the pursuit for a cure. This paper presents
an overview on microalgae with anti-cancer activities. Microalgae are eukaryotic unicellular plants
that contribute up to 40% of global primary productivity. They are excellent sources of pigments,
lipids, carotenoids, omega-3 fatty acids, polysaccharides, vitamins and other fine chemicals, and there
is an increasing demand for their use as nutraceuticals and food supplements. Some microalgae
are also reported as having anti-cancer activity. In this review, we report the microalgal species that
have shown anti-cancer properties, the cancer cell lines affected by algae and the concentrations of
compounds/extracts tested to induce arrest of cell growth. We also report the mediums used for
growing microalgae that showed anti-cancer activity and compare the bioactivity of these microalgae
with marine anticancer drugs already on the market and in phase III clinical trials. Finally, we discuss
why some microalgae can be promising sources of anti-cancer compounds for future development.
Keywords: marine biotechnology; microalgae; anti-cancer
1. Introduction
Cancer includes a large group of pathologies related to the unrestrained proliferation of cells in
the body [1]. There are more than 200 different types of cancers, and some cancers may eventually
spread into other tissues causing metastases that are often lethal. Cancer is the leading cause of death
globally, largely due to aging and growth of the world’s population. According to the European
Cancer Observatory [2], estimates for the four most common types of cancer in the European Union in
2012 were as follows: 342,137 cases of colon cancer, 309,589 cases of lung cancer (including trachea
and bronchus cancer), 358,967 cases of breast cancer and 82,075 cases of skin melanoma. Finding
more effective methods to treat cancer remains a challenge, and development of new therapeutic
agents for cancer treatment is essential for continued progress against the disease. According to
Dyshlovoy and Honecker [3] approximately 60% of the drugs used in hematology and oncology
have their origin in natural sources, and one third of the most sold are either natural compounds or
derivatives thereof. There has also been growing interest in marine bioprospecting, because potent
natural compounds (e.g., terpenes, steroids, alkaloids, polyketides, etc.) have already been discovered
from marine organisms. Currently there are seven drugs of marine origin on the market, four of
which are anticancer drugs. There are also close to 26 marine natural products in clinical trials of
which 23 are anti-cancer compounds [4]. Oceans cover nearly 70% of the planet, but remain largely
unexplored. To date, more than 28,000 compounds isolated from marine organisms have been reported,
and this number is rapidly growing each year [4]. However, despite the number of compounds isolated
from marine organisms and the biological activities attributed to many of these, the search for ocean
medicines is relatively recent and only in the middle part of the 20th century did scientists begin to
systematically probe the oceans for new drugs. Today, the pipeline from the initial demonstration that
Mar. Drugs 2018, 16, 165; doi:10.3390/md16050165 www.mdpi.com/journal/marinedrugsMar. Drugs 2018, 16, 165 2 of 17
a molecule may have therapeutic potential to the production of an approved drug involves pre-clinical
testing, complex clinical trials in humans, and post-trial regulatory approval by the Food and Drug
Administration (FDA). For drugs, this process can take 10 to 15 years (Figure 1) and costs millions of
dollars [5], with less than 12% of the potential drugs receiving final approval [6].
Figure 1. Time estimates for research and development of new Food and Drug Administration (FDA)
approved drugs.
Several factors, such as difficulties in harvesting organisms, low quantities of active compounds in
extracts, finding adequate procedures for isolation and purification, possible toxicity of the compounds
and sustainable production of compounds may slow down the entire pipeline. Notwithstanding these
difficulties, the discovery of new ocean medicines is one of the most promising new directions of marine
science today. Novel initiatives with marine organisms aimed at enabling environmentally-friendly
approaches to drug discovery have been tackled in several European Union 7th Framework Programme
(EU FP7) and European Union Horizon 2020 projects (EU H2020), such as Biologically Active
Molecules of Marine Based Origin (BAMMBO), Bluegenics, European Marine Biological Research
Infrastructure Cluster (EMBRIC), Genetic Improvement of Algae for Value Added Products (GIAVAP),
Marine Microorganisms Cultivation Methods for Improving their Biotechnological Applications
(MaCuMBA) and PharmaSea. This has led to several technological advancements in culturing micro-
and macro-organisms, increased sampling efforts in diverse and often extreme habitats that have led to
the discovery of species that are new for science, massive sequencing of genomes and transcriptomes
allowing for the identification of new metabolic pathways and/or assignment of potential functions
to unknown genes. Here, we discuss the marine microalgae which have shown anti-cancer activity.
This is the first review on this subject because recent studies have indicated that microalgae may
represent a reservoir for new bioactive compounds that can act as anti-cancer drugs.
2. Marine Microalgae
Microalgae are eukaryotic plants that contribute up to 40% of global productivity [7]. They are
at the base of aquatic food webs, have short generation times (doubling time = 5–8 h for some
species) and have colonized almost all biotopes, from temperate to extreme environments (e.g., cold
environments and hydrothermal vents). Their advantage in marine drug discovery is their metabolic
plasticity, which can trigger the production of several compounds with possible applications in various
biotechnology sectors (i.e., food, energy, health, environment and biomaterials) [8,9]. They can beMar. Drugs 2018, 16, 165 3 of 17
easily cultivated in photo-bioreactors (e.g., in 100,000 L bioreactors) to obtain a huge biomass and
represent a renewable and still poorly-explored resource for drug discovery. They use solar energy
and fix CO2 which contributes to the mitigation of greenhouse gas effects and the removal of nitrogen
and phosphorous derivatives which can be pollutants depending on their concentration [10]. Table 1
reports the microalgal species that have shown anti-cancer properties, the cancer cell lines affected by
microalgae and the concentrations that have been tested to induce the arrest of cell growth.Mar. Drugs 2018, 16, 165 4 of 17
Table 1. Active microalgal species, active fraction/compounds tested and cell lines against which these have proven to be effective (CV stands for cell viability).
Microalgae Fraction/Compound Target Cells Active Concentration Reference
Thalassiosira rotula, Skeletonema costatum
and Pseudonitzschia delicatissima.
Commercial source, not
from microalgae
Polyunsaturated aldehydes (PUAs)
Colon adenocarcinoma (Caco-2)
Lung adenocarcinoma (A549)
Colon adenocarcinoma (COLO 205)
11 to 17 µg/mL (arrest of cell growth)
0.22 to 1.5 µg/mL (CV of 80% to 0% depending on
the conditions)
[11]
[12]
Chlorella ellipsoidea Carotenoid extract Colon carcinoma (HCT-116) 40 µg/mL (IC50) [13]
Synedra acus Chrysolaminaran (polysaccharide) Colorectal adenocarcinoma (HT-29 and DLD-1) 54.5 and 47.7 µg/mL (IC50 for HT-29 and DLD-1) [14]
Dunaliella tertiolecta Violaxanthin (carotenoid already
identified in C. ellipsoidea) Breast adenocarcinoma (MCF-7) 40 µg/mL (to observe cytostatic activity) [15]
Cocconeis scutellum Eicosapentaenoic
acid (EPA) Breast carcinoma (BT20) Not clarified [16]
Chaetoseros sp., Cylinrotheca closterium,
Odontella aurita and
Phaeodactylum tricornutum
Fucoxanthin (carotenoid)
Promyelocytic leukemia (HL-60), Caco-2, colon
adenocarcinoma (HT-29), DLD-1 and prostate
cancer (PC-3, DU145 and LNCaP)
29.78 µg/mL (CV of 17.3% for HL-60)
10.01 µg/mL (CV of 14.8%, 29.4% and 50.8% for
Caco-2, DLD-1 and HT-29)
13.18 µg/mL (CV of 14.9%, 5.0% and 9.8% for
PC-3, DU145 and LNCaP)
[17]
Chaetoceros calcitrans EtOH extract
AcOEt extract
MCF-7
Breast adenocarcinoma (MDA-MB-231)
3.00 µg/mL (IC50)
60 µg/mL (IC50)
[18]
[19]
Amphidinium carterae
CH3Cl fraction
Hexane fraction
AcOEt fraction
HL-60
HL60, Skin melanoma (B16F10), A549
50 µg/mL (CV of 40%)
25–50 µg/mL (CV between 50% and 90%) [20]
Eleven strains of benthic diatoms
Ostreopsis ovata
Amphidinium operculatum
MeOH extract HL-60 50 µg/mL (CV of 48% for O. ovata and 58% for A.
operculatumi) [21]
Navicula incerta Stigmasterol (phytosterol) Liver hepatocellular carcinoma (HepG2) 8.25 µg/mL (CV of 54%) [22]
Phaeodactylum tricornutum
Nonyl-8-acetoxy-6-methyloctanoate
(NAMO, fatty alcohol ester)
Monogalactosyl glycerols 1
HL-60
Mouse epithelial cell lines (W2, D3)
22.3 µg/mL (IC50)
40-50 µg/mL (concentration necessary to induce
apoptosis)
[23]
[24]
Skeletonema costatum
Skeletonema marinoi
Hydrophobic fraction and PUAs
Hydrophobic fraction
Caco-2
(A2058 not affected)
Skin melanoma (A2058)
11 to 17 µg/mL (PUAs)
50 µg/mL (CV of 60%)
[11]
[8]
Canadian marine microalgal pool Aqueous extract
A549, lung carcinoma (H460), prostate carcinoma
(PC-3, DU145), stomach carcinoma (N87), MCF-7,
pancreas adenocarcinoma (BxPC-3) and
osteosarcoma (MNNG)
5000 µg/mL (CV between 30% and 80%
depending on the cell line) [25]
Chlorella sorokiniana Aqueous extract A549 and lung adenocarcinoma (CL1-5) 0.0156 to 1 µg/mL (CV reduced down to
20% progressively) [26]
1 (2S)-1-O-5,8,11,14,17-eicosapentaenoyl-2-O-6,9,12-hexadecatrienoyl-3-O-[β-D-galactopyranosyl]-glycerol and (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-2-O 6,9,12,15-octadecatetraenoyl-3-
O-β-D-galactopyranosyl-sn-glycerol.Mar. Drugs 2018, 16, 165 5 of 17
As reported in previous studies, the bioactivity of microalgae may differ for different clones and can
vary depending on the culturing conditions (e.g., nutrient availability, temperature, light intensity) [8,27]
and growth phase [28]. For example, Ingebrigtsen et al. [27] demonstrated that the bioactivity of various
marine microalgae extracts (i.e., the diatoms Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus,
Skeletonema marinoi and Porosira glacialis) with anti-cancer activity against melanoma A2058 cells changed
when they were cultured under different light and temperature conditions. Lauritano et al. [8] also
showed that microalgal bioactivity can vary depending on the nutrient concentrations used for their
cultivation. These authors showed that the diatom Skeletonema marinoi had anti-cancer activity exclusively
when cultured under nitrogen starvation conditions. Considering the importance of culturing conditions
(Table 2), we report the mediums used for growing marine microalgae that showed anti-cancer activity
(e.g., Conway’s medium, Guillard’s F/2 medium or variations of both mediums) and, where available,
the sampling locations and harvesting times.
Table 2. Active microalgal species, sources, culturing conditions and references.
Microalgae Source Culturing Conditions Harvesting Time Reference
Synedra acus Lake Baikal
Culture medium consisting of (mg/L)
Ca(NO3)2·4H2O (20), KH2PO4 (2), MgSO4 (12),
NaHCO3 (30), Na2EDTA (2.2), H3BO3 (2.4),
MnCl2·4H2O (1.3), (NH4)6Mo7O24·4H2O (1),
Na2SiO3·9H2O (25), FeCl3 (1.6), cyanocobalamine
(0.04), thiamine (0.04), and biotin (0.04).
12 ◦C and 250–300 µmol·m−2
·s−1 light intensity.
Not provided [14]
Dunaliella tertiolecta DT strain CCMP364
(NCMA, USA)
Conway medium.
20 ◦C, 180 µmol·m−2
·s−1 light intensity.
Late exponential
phase [15]
Cocconeis scutellum Mediterranean Sea, Stazione
Zoologica A. Dohrn
Guillard’s F/2 medium.18◦C, 140 µmol·m−2
·s−1
light intensity and 12 h:12 h photoperiod. Not provided [16]
Chaetoceros
calcitrans
Strain UPMAAHU10
University Putra Malaysi
Conway medium.
24 ◦C, 120 µmol·m−2
·s−1 light intensity, automatic
oscillating shaker at 110 rpm and harvested at
stationary phase (6–7 days).
Conway medium.
Conditions not provided.
Stationary phase
Not provided
[18]
[19]
Amphidinium
carterae
Korea Marine Microalgae
Culture Center
Conway medium.
20 ◦C, 34 µmol·m−2
·s−1 light intensity and 24 h:0 h
photoperiod.
Days 8–10 [20]
Eleven strains of
benthic dinoflagellates Coast of Jeju Island (Korea)
Daigo IMK medium (Nihon Pharmaceutical Co.,
Ltd.) and Guillard’s F/2 medium.
20 ◦C, 180 µmol·m−2
·s−1 light intensity and 12 h:12
h photoperiod.
Exponential phase. [21]
Navicula incerta Korea Marine Microalgae
Culture Center.
Guillard’s F/2 medium.
Conditions not provided. Not provided [22]
Phaeodactylum
tricornutum
Korea Marine Microalgae
Culture Center
Provasoli-Guillard
National Center
Conway medium.
20 ◦C, 34 µmol·m−2
·s−1 light intensity and 24 h:0 h
photoperiod.
Guillard’s F/2 medium.
18 ◦C and 100 µmol·m−2
·s−1 light intensity.
Days 8–10
Not provided
[23]
[24]
Skeletonema marinoi
FE6 (1997)
FE60 (2005)
Adriatic Sea
(Mediterranean Sea)
Guillard’s F/2 medium.
19 ◦C, 100 µmol·m−2
·s−1 light intensity and 12 h:12
h photoperiod.
Late stationary
phase [8]
3. Active Fractions from Marine Microalgae
3.1. Carotenoid Extract from Chlorella Ellipsoidea
Chlorella species are widely known as being a good commercial source of carotenoids such as lutein,
β-carotene, zeaxanthin and astaxanthin. Kwang et al. 2008 [13] tested the anti-proliferative effect of the
carotenoids extracted from the green algae C. ellipsoidea and C. vulgaris on a human colon carcinoma cell
line (HCT116). Briefly, a freeze-dried Chlorella powder was extracted with ethanol, treated with a solution
of KOH for saponification and further partitioned with hexane. This hexane phase, rich in carotenoids, was
analyzed using HPLC-ESI-MS in order to identify the major carotenoid composition. They found that the
carotenoid extract of C. ellipsoidea was mainly composed of violaxanthin and, in lower ratios, by two other
xanthophylls (antheraxanthin and zeaxanthin). The extract from C. vulgaris was composed mainly ofMar. Drugs 2018, 16, 165 6 of 17
lutein. Anti-cancer activity was measured using the MTT assay after 24 h of exposure with the microalgal
extracts. The half maximal inhibitory concentration (IC50) value was 40.73 ± 3.71 µg/mL for C. ellipsoidea
and 40.31 ± 4.43 µg/mL for C. vulgaris—much higher than the IC50 of pure lutein (21.02 ± 0.85 µg/mL).
In order to understand if apoptosis is linked to the observed anti-proliferative effect, the authors also
performed an annexin V-fluorescein assay to check phosphatidylserine translocation (index of apoptosis).
The apoptotic effect was confirmed after treatment with both C. ellipsoidea and C. vulgaris extracts.
They reported that apoptosis was 2.5-fold higher in the case of the C. ellipsoidea extract. The carotenoid
extract was not tested on normal human cell lines.
3.2. Ethanol and Ethyl Acetate Extracts from Chaetoceros Calcitrans
Nigjeh et al. [18] tested the ethanolic extract from the planktonic diatom Chaetoceros calcitrans on
breast adenocarcinoma (MCF-7), breast epithelial (MCF-10A) and peripheral blood mononuclear cells
(PMBC). The ethanolic extract was obtained after the homogenization of the microalgal biomass with
absolute ethanol and further filtration of the supernatant using filter cotton and a 0.2 µm filtration
unit. The results were compared with the effects of tamoxifen, an already known drug used for the
treatment of breast cancer. Cell viability was performed using the MTT assay on MCF-7, MCF-10A and
PMBC cells. The results were expressed as IC50 values obtained by screening different concentrations
(0 to 30 µg/mL) of C. calcitrans extract for 24 and 72 h. The IC50 values of the ethanolic extract screened
on MCF-7 were 3.00 ± 0.65 µg/mL for 24 h and 2.69 ± 0.24 µg/mL for 72 h, while the IC50 values
of MCF-10A cells were 12.00 ± 0.59 µg/mL for 24 h and 3.30 ± 0.36 µg/mL for 72 h. C. calcitrans
extract did not display any cytotoxicity on PMBC cells even at the highest concentrations; the activity
was specific to the cancer cells. The IC50 of tamoxifen on MCF-7 was 12.00 ± 0.52 µg/mL for 24 h
and 9.00 ± 0.40 µg/mL for 72 h. The comparison with tamoxifen shows that the anti-cancer activity
of C. calcitrans extract is very interesting. In addition, annexin V/propidium iodide analyses were
performed and the results indicated apoptosis induction in MCF-7 cells after treatment with the
extract. The authors also observed an increase in the proapoptotic protein Bax, and the caspases 3 and
7 transcripts.
Goh et al. [19] analyzed the effects of the extracts from C. calcitrans against a wide range
of cancer cell lines. In particular, they studied the cytotoxicity of four crude solvent extracts
(hexane, dichloromethane, ethyl acetate and methanol) in the following cancer cell lines: human
breast adenocarcinoma (MDA-MB-231), MCF-7, mouse breast carcinoma (4T1), liver hepatocellular
carcinoma (HepG2), cervix epithelial carcinoma (HeLa), human prostate carcinoma (PC-3), human lung
adenocarcinoma (A549), human colon adenocarcinoma (HT-29), and human ovarian adenocarcinoma
(Caov3). A mouse embryo fibroblast (3T3) cell line was used to measure cytotoxicity against
non-tumorigenic cells. A freeze-dried powder of C. calcitrans was shaken in hexane, dichloromethane,
ethyl acetate or methanol (MeOH) for 24 h and filtered through cotton to obtain the different extracts.
The MTT assay was carried out after 72 h of treatment with the microalgal extracts and doxorubicin
was used as a control (60 µg/mL of doxorubicin). The authors observed that crude ethyl acetate extract
from C. calcitrans had cytotoxic properties in the MDA-MB-231 cancer cell line with IC50 of 60 µg/mL.
An assay on a non-tumorigenic fibroblast cell line also revealed that the cytotoxic effect was specific
against cancer cells; the extract did not have cytotoxic effects on the 3T3 cell line.
3.3. Organic Fractions from Amphidinium Carterae
Samarakoon et al. [20] tested the anti-proliferative activity of various fractions from the
dinoflagellate Amphidinium carterae extract on different cancer cell lines: HL-60 (Human promyelocytic
leukemia cells), B16F10 (mouse melanoma tumor cells), and A549 (adenocarcinomic human alveolar
basal epithelial cells). Cytotoxicity assays were also carried out using the mouse monocyte macrophage
cell line (RAW 264.7). Freeze-dried biomass from the cultured marine microalgae was grounded into
fine powder, extracted with methanol (80%) and homogenized by sonication at 25◦C for 90 min.
The crude methanol extract was concentrated by evaporating the solvent under reduced pressure usingMar. Drugs 2018, 16, 165 7 of 17
a rotary evaporator and further partitioned. Analytical grade n-hexane, chloroform, ethyl acetate,
and water were used in solvent-solvent partition chromatography in order to obtain the fractions to be
tested. Cell growth inhibition was measured with the MTT assay. A. carterae chloroform fraction was
the most active and reduced HL-60 cell viability by about 50% after 24 h exposure at a concentration of
50 µg/mL. No tests were performed on normal human cell lines.
3.4. Methanolic Extracts from Amphidinium Carterae, Prorocentrum Rhathymum, Symbiodinium sp., Coolia
Malayensis, Ostreopsis Ovata, Amphidinium operculatum and Heterocapsa psammophila
Shah et al. [21] cultivated up to eleven different strains of benthic dinoflagellates (Amphidinium carterae,
Prorocentrum rhathymum, Symbiodinium sp., Coolia malayensis strain 1, Ostreopsis ovata strain 1, Ostreopsis
ovata strain 2, Coolia malayensis strain 2, Amphidinium operculatum strain 1, Heterocapsa psammophila, Coolia
malayensis strain 3 and Amphidinium operculatum strain 2) isolated from the coast of Jeju Island (Korea) in
2011, to screen on RAW 264.7 (murine macrophage cell line) and HL-60 (human promyelocytic leukemia
cell line) cells. They specified the specific sampling location and the microalgal growth phase tested
(Table 2). To obtain the methanolic extracts, the freeze-dried biomass from the cultured marine microalgae
was ground into fine powder, extracted with methanol (80%) and homogenized by sonication at 25◦C
for 90 min. An MTT assay was carried out to study cell viability after extract exposure for 24 h at 37◦C.
In this case, only Ostreopsis ovata 1 and Amphidinium operculatum 1 significantly inhibited the growth of
HL-60 cancer cells (reducing cell viability between 40 and 60% compared to the control, at a concentration
of 50 µg/mL). No tests were performed on normal human cell lines.
3.5. Hydrophobic Fraction from Skeletonema Marinoi
Lauritano et al. [8] studied the effects of 32 species of microalgae identified by microscopy and 18S
sequencing. The microalgal biomass from the cultured marine microalgae was extracted with a ratio of
acetone:water (1:1) and further fractionated using Amberlite RXAD16N resin with acetone as the resin
eluent to obtain an hydrophobic fraction. The authors found that hydrophobic fractions from Skeletonema
marinoi, Alexandrium minutum, Alexandrium tamutum and Alexandrium andersoni were active against
a melanoma cancer cell line (A2058) at a concentration of 100 µg/mL. Further testing on a normal lung
fibroblast (MRC-5) cell line showed that Alexandrium species were toxic. Two different strains of Skeletonema
marinoi (FE6 and FE60 strains from the Adriatic Sea) were tested on an A2058 cell line. The colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, a tetrazolium
dye used for the quantification of viable cells) assay was performed to check the cytotoxicity in both normal
and cancer cell lines. The results showed that only the FE60 strain was active against A2058 and only when
cultured under nitrogen-starvation conditions. FE60 reduced the cell viability to 60% when screened at
50 µg/mL and to 10% when screened at 100 µg/mL.
3.6. Aqueous Extract from a Canadian Marine Microalgal Pool
Somasekharan et al. [25] studied the anti-proliferative effect of a raw marine microalgal material
from Canada (dried powder) on eight different cancer cell lines, together with the anti-colony
forming activity in the same lines. The dried microalgal powder was suspended in distilled water at
a concentration of 30 mg/mL and then sonicated with short bursts. The sonicated suspension was
then passed through a 25-gauge needle to release the cytosolic contents, followed by syringe filtration
through 0.2 µm filters. This aqueous extract from the raw material was tested on A549, H460 (lung
adenocarcinoma cell lines), PC-3, DU145 (prostate cancer cell lines), N87 (stomach cancer cell line),
MCF7 (breast cancer cell line), BxPC-3 (pancreas cancer cell line) and MNNG (bone cancer cell line).
The cells were incubated with the extract for 72 h at 0 (control), 1, 2 and 5 mg/mL. Cell viability was
determined using the MTT assay. The extract did not show any significant activity at 1–2 mg/mL except
for on the MNNG cell line (50% reduction in cell viability at 2 mg/mL). At 5 mg/mL proliferation
of almost all the cell lines were significantly inhibited. Tests on normal human cell lines were not
performed. The authors performed a crystal violet test at 0.5–5 mg/mL to check the anti-colony activityMar. Drugs 2018, 16, 165 8 of 17
of the extract. This test indicated that the extract successfully inhibited the colony forming ability of all
cancer cells tested even at the lowest concentration (0.5 mg/mL).
3.7. Aqueous Extract from Chlorella Sorokiniana
Chlorella sp. biomass is widely used as a dietary supplement in many countries and is mostly
produced in Asia (https://www.marketresearchfuture.com/reports/chlorella-market-4413). There are
also some patents related to its use as a dietary supplement (e.g., US 2005/0196389 A1 [29]). Lin et al. [26]
studied the effects of hot water extracts from the diatom Chlorella sorokiniana (marine strain) on lung
adenocarcinoma cell lines (A549 and CL1-5). The extracts were obtained by reflux extraction of the
dried biomass with distilled water for 1 h and further filtration with N0.5 filter paper. To determine the
cytotoxicity, the authors performed the MTT assay at a concentration range of 15.625 to 1000 ng/mL;
the results indicated a dose-dependent reduction in cell viability on both cancer cell lines. The cytotoxicity
on normal human cells was not tested. They also studied the mechanism of action of C. sorokiniana extract
using annexin V/propidium iodide staining (flow cytometry analysis) to confirm a possible cell cycle
arrest/apoptotic process. Cell cycle arrest was not observed even after 24 h of exposure, but an increment
in the number of cells in sub-G1 phase was observed, which is a phenomenon that typically indicates
apoptosis. Protein expression (Western blot analysis) of the cleaved and activated forms of caspase
9, caspase 3 and Poly (ADP-ribose) polymerase (PARP) increased in both cell lines after exposure to
the microalgal extract after 24 h. The activation of caspase 9 and caspase 3 suggested that the main
pathway involved in apoptosis was the mitochondrial pathway. In addition, the ratio of Bax/Bcl-2
(pro/antiapoptotic proteins) increased after 24 h of treatment which is another sign of apoptosis.
4. Active Compounds from Marine Microalgae
4.1. Polyunsaturated Aldehides (PUAs)
Miralto et al. [11] isolated three polyunsaturated aldehydes (PUAs, Figure 2) from the marine
diatoms Thalassiosira rotula, S. costatum and P. delicatissima. They found that 2-trans-4-cis-7-cis-
decatrienal, 2-trans-4-trans-7-cis-decatrienal and 2-trans-4-trans-decadienal had anti-proliferative
activity on the human colon adenocarcinoma cell line (Caco-2). To check the anti-proliferative
activity, they used different concentrations of PUAs between 0 and 20 µg/mL after 48 h of incubation.
Concentrations of 11–17 µg/mL were enough to reduced cell viability to almost 0%. In addition,
a TUNEL assay was performed to check DNA fragmentation and to verify that apoptosis had occurred.
Figure 2. Polyunsaturated aldehydes. From left to right: 2-trans-4-cis-7-cis-decatrienal (a);
2-trans-4-trans-7-cis-decatrienal (b); 2-trans-4-trans-decadienal (c); 2-trans-4-trans-octadienal (d) and
2-trans-4-trans-heptadienal (e).
Sansone et al. [12] tested the effect of the commercially-available PUAs 2-trans-4-trans-decadienal
(DD), 2-trans-4-trans-octadienal (OD) and 2-trans-4-trans-heptadienal (HD) (Figure 2) on the
adenocarcinoma cell lines, lung A549 and colon COLO 205. The authors tested these three
polyunsaturated aldehydes at different exposure times (i.e., 48 and 72 h) and concentrations (i.e., 2,
5 and 10 µM). For quantities of 2, 5 and 10 µM DD decreases in cell viability of 70%, 50% and 18%
respectively, were induced in A549 cells after 24 h. For the COLO 205 cell line, cell viability decreased
to 80%, 44% and 26% with 2, 5 and 10 µM DD, respectively. In the case of OD, 10 µM of the compoundMar. Drugs 2018, 16, 165 9 of 17
decreased cell viability to 35% after 72 h in A549 cells. At 2, 5 and 10 µM, OD also reduced cell viability
to 60%, 60% and 41% in COLO 205 cells, respectively, after 72 h. At 10 µM, HD reduced cell viability to
10% in A549 cells after 48 h and 0% after 72 h, while the same concentration tested on COLO 205 cells
reduced cell viability to 40% after 48 h and 28% after 72 h. The authors also tested the three PUAs on
a normal lung/brunch epithelial BEAS-2B cell line to check cytotoxicity on normal cells. None of the
PUAs were toxic.
4.2. Chrysolaminaran Polysaccharide
Kusaikin et al. [14] isolated one polysaccharide of the chrysolaminaran family (Figure 3) from
the diatom Synedra acus. These storage polysaccharides are well known to be common water soluble
biopolymers synthetized by diatoms [30]. The anti-tumor activity of the chrysolaminaran extracted
from S. acus was studied on HTC-116 and DLD-1 human colon cancer cell lines. The MTS method
was carried out to determine cell viability. Cancer cells were treated with 25, 50 and 100 µg/mL of
chrysolaminaran for up to 72 h. The inhibition trend in the different experiments was irregular, but the
IC50 values were determined for each cell line: 54.5 µg/mL for HCT-116 and 47.7 µg/mL for DLD-1.
The authors did not find any toxicity on the HTC-116 and DLD-1 cell lines at concentrations above
200 mg/mL and, considering that most anti-tumor drugs are toxic at these concentrations, this is a very
promising property. The degree of cytotoxicity on normal human cells was not tested.
Figure 3. Chrysolaminaran monomer.
4.3. Violaxanthin
Pasquet et al. [15] performed an anti-cancer screening of extracts from the green algae
Dunaliella tertiolecta on four different cancer cell lines: MCF-7, MDA-MB-231, A549 and LNCaP.
They prepared different extracts using a wide range of solvents in terms of polarity, including
dichloromethane, ethanol and ultrapure water. The MTT assay was used to evaluate the cells’ viability.
The dichloromethane extract showed significant activity against MCF-7 cancer cells. RP-HPLC analysis
and fractionation was used to obtain one subfraction of the dichloromethane extract that was also
screened for 72 h at concentrations between 0.1 µg/mL and 40 µg/mL. The subfraction was then
identified as violaxanthin (Figure 4) at a rate of 95%. In addition to these results, the DNA of non-treated
and treated cells was extracted and analysed using standard electrophoresis. Despite indications of
early apoptosis (phosphatidylserines translocation detected using annexin-V-Alexa 568 fluorochrome),
the violaxanthin subfraction did not cause any DNA fragmentation. Cytotoxicity tests were not
performed in normal human cell lines.
Figure 4. Violaxanthin.Mar. Drugs 2018, 16, 165 10 of 17
4.4. Eicosapentaenoic Acid (EPA)
Nappo et al. [16] screened the extracts from the marine diatom Cocconeis scutellum on the following
cells lines: BT20 (human breast cancer), MB-MDA468 (human breast cancer), LNCaP (human prostate
adenocarcinoma cells), COR (Epstein-Barr Virus-transformed B cells isolated from human tonsils),
JVM2 (lymphoblast immortalized with Epstein-Barr virus) and BRG-M (Burkitt’s lymphoma cells).
The results of the screening were not entirely published but the authors determined that C. scutellum
extract was more effective on the BT20 cell line. The degree of cytotoxicity normal human cell lines was
not studied. The fractionation of diethyl ether extract (the most active) from C. scutellum produced three
fractions with differentiated activities. Fractions 1–2 did not induce any significant reduction in cell
viability compared to the control but fraction 3 reduced the viability to 56.2%. DNA fragmentation was
evaluated with the annexin V/propidium iodide staining methods. The analysis of the composition of
these fractions indicated that fraction 1 contained glycerides (77.2% of total ion current) and fatty acids
(2.4%), fraction 2 contained fatty acids (66.7%), monoglycerides (11.0%) and sterols (3.2%), and finally,
fraction 3 contained fatty acids (81.7%) and 4-methylcholesterol (2.3%). The authors concluded that
the fatty acid subfractions were responsible for this activity, specifically, eicosapentaenoic acid (EPA,
Figure 5). This conclusion was reached because EPA was the only product in the fraction that has been
reported to induce apoptosis [31]. The activation of caspases 8 and 3 was also confirmed by Western
blot analysis. The authors concluded that it is not yet clear whether EPA is the only factor involved
in the apoptosis of BT20 cells or if there is a synergic association among different compounds in the
same fraction.
Figure 5. Eicosapentaenoic acid.
4.5. Fucoxanthin
Fucoxanthin (Figure 6) is one of the most studied compounds that can be found in marine micro-
and macroalgae. It is a pigment from the family of the xanthophylls and a major carotenoid in brown
algae [32]. Kadekaru et al. [33] evaluated fucoxanthin toxicity by providing oral doses (10 mg/kg
and 50 mg/kg) to rats for a period of 28 days. Fucoxanthin did not show any obvious toxicity and
is hence considered safe as a pharmaceutical ingredient. Ishikawa et al. [34] performed a similar
analysis in mice using a metabolite of fucoxanthin, fucoxanthinol. In this case, they used a higher dose
(200 mg/kg) for 28 days, but it did not show any toxicity either.
Hosokawa et al. [35] showed that fucoxanthin had strong anti-proliferative activity against HL-60
cells and could also induce apoptosis. Cells treated with 11.3 and 45.2 µM of fucoxanthin showed
viabilities of 46.0% and 17.3%, respectively, after 24 h. Cell viability was determined by the dye
exclusion test using trypan blue. Hosokawa et al. 2004 also tested the cell viability on three human
colon cancer cell lines (Caco-2, DLD-1 and HT-29) treated with fucoxanthin. Caco-2, DLD-1 and HT-29
cells showed a dose-time dependent trend. The Caco-2 cell line was more affected than the other two
cell lines (analyzed by WST-1 assay). Normal human cells were not tested.
Kotake-Nara et al. [36] examined 15 types of carotenoids (Neoxanthin, fucoxanthin, phytofluene,
lycopene, phytoene, canthaxanthin, β-cryptoxanthin, zeaxanthin, β-carotene, α-carotene, γ-carotene,
astaxanthin, capsanthin, lutein and violaxanthin) on three different prostate cancer cell lines (PC-3,
DU145 and LNCaP) and found that fucoxanthin was one of the most active anti-cancer compounds.Mar. Drugs 2018, 16, 165 11 of 17
The percentages of viable cells after 72 h when fucoxanthin was added at 20 µM were 14.9% for PC-3,
5.0% for DU145 and 9.8% for LNCaP, respectively (determined by MTT assay). Normal human cells
were not tested.
Peng et al. [17] summarized the studies related to fucoxanthin, the microalgae that are known to
produce it (i.e., Chaetoseros sp., Cylinrotheca closterium, Odontella aurita and Phaeodactylum tricornutum)
and its role as a bioactive compound (e.g., as an antioxidant, anti-inflamatory, anti-cancer, anti-diabetic,
skin protective agent, bone protective agent, etc.).
Figure 6. Fucoxanthin.
4.6. Stigmasterol
Kim et al. [22] isolated Stigmasterol (Figure 7) from Navicula incerta extracts using chromatography
techniques such as silica gel open column chromatography and preparative thin layer chromatography
(PTLC). They screened the anti-proliferative effect of the isolated stigmasterol at 5, 10 and 20 µM
on HepG2 (human liver cancer cell line). Cytotoxicity values of 40%, 43% and 54% were found,
respectively, which indicated a dose-dependent trend. The cytotoxic effects on normal human cells
were not studied. The phytosterol-like structures with double bonds in the C-5 and C-22 positions,
like stigmasterol, have been shown to induce apoptosis [37]. In this case, apoptosis was studied by
controlling morphological changes, fluorescence-activated cell sorting, apoptosis pathways analysis,
gene expression levels and also, with flow cytometric measurement of cell cycle arrest. All these
assays indicated that stigmasterol has a huge apoptosis induction capability, probably via an apoptosis
signaling pathway in the mitochondria.
Figure 7. Stigmasterol.
4.7. NAMO (Nonyl 8-acetoxy-6-methyloctanoate)
Samarakoon et al. [23] tested the anti-cancer activity of 8-acetoxy-6-methyloctanoate (NAMO,
Figure 8) obtained from Phaeodactylum tricornutum against three different cell lines: human
promyelocytic leukemia cell line (HL-60), a human lung carcinoma cell line (A549) and a mouse
melanoma cell line (B16F10). NAMO was screened at 25 and 50 µg/mL for 48 h. NAMO was only
active against HL-60 cells at both concentrations tested. The highest growth inhibitory activity of
about 70% on HL-60 cells was observed at a concentration of 50 µg/mL NAMO. The cytotoxic effects
on normal human cells were not studied. Regarding its mechanism of action, NAMO induced DNA
damage and increased apoptotic body formation. Cell cycle arrest and the accumulation of cells in the
sub-G1 phase were observed to occur proportionally to the concentration of NAMO. The authors alsoMar. Drugs 2018, 16, 165 12 of 17
observed activation of the pro-apoptotic protein Bax, suppression of the anti-apoptotic protein Bcl-x,
and an increase in the expression of both caspase-3 and p53 proteins.
Figure 8. Nonyl 8-acetoxy-6-methyloctanoate (NAMO).
4.8. Monogalactosyl Glycerols
Andrianasolo et al. [24] isolated two different monogalactosyl glycerols (Figures 9 and 10) from
Phaeodactylum tricornutum and tested them against immortal mouse epithelial cells (W2 and D3). The W2
cell line is a wild type, while D3 cells have the apoptosis function disabled through gene deletion
(this assay is one of the approaches for the study of apoptosis and its role in cancer and oncogenesis).
The minimum values required for apoptosis induction with this test were a death rate of 20% on the
W2 cell line and, at a growth rate of at least 10% on the D3 cell line. For compound 1 (Figure 9) (52 µM)
the W2 death rate was 18% ± 1% and the D3 growth rate was 10% ± 1%. For compound 2 (Figure 10)
(64 µM), the W2 death rate was 18% ± 1% and the D3 growth rate was 14% ± 1%. The results confirmed
that the isolated compounds have specific apoptosis activity against the W2 cell line.
Figure 9. Monogalactosyl Glycerol (Compound 1): (2S)-1-O-5,8,11,14,17-eicosapentaenoyl-2-O-6,9,12-
hexadecatrienoyl-3-O-[β-D-galactopyranosyl]-glycerol.
Figure 10. Monogalactosyl Glycerol (Compound 2): (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-2-O-6,9,12,15-
octadecatetraenoyl-3-O-β-D-galactopyranosyl-sn-glycerol.
5. Active Compounds from Other Marine Organisms
To better understand the potential of microalgal species as important sources of anti-cancer
compounds, we compared their bioactivity with marine anti-cancer drugs already on the market.
Tables 3 and 4 report the anti-cancer compounds already on the market and in phase III clinical trials,
the marine organisms from which the compounds were isolated, the active compounds, the target
cancer cell lines and the active concentrations that arrest the growth of cancer cells.Mar. Drugs 2018, 16, 165 13 of 17
Table 3. Active marine-derived compounds available on the market. The table reports the producing
marine organisms, the active compounds, the target cancer cell lines, the active concentrations
and references.
Marine Organism Compound Target Active Concentration Reference
Ecteinascidia
turbinata
Ecteinascidin/
Trabectedin (alkaloid)
MFC7
A549
0.6 ng/mL (IC70)
5.6 ng/mL (IC70) [38]
Dolabella
auricularia/Symploca
sp. VP642
Brentuximab vedotin
(antibody-drug conjugate)
Non-Hodgkin’s lymphoma
cells (Karpas 299) 2.5 ng/mL (IC50) [39]
Halichondria okadai Eribulin mesylate
(macrolide)
DLD-1
LNCaP
HL-60
6.934 ng/mL (IC50)
0.365 ng/mL (IC50)
0.657 ng/mL (IC50)
[40]
Cryptotheca crypta Cytarabine (nucleoside) Acute Myeloid Leukemia
(AML) cells 272 ng/mL (IC50) [41]
Table 4. Active marine-derived compounds in phase III clinical trials. The table reports the producing
marine organisms, the active compounds, the target cancer cell lines, the active concentrations
and references.
Marine Organism Compound Target Active Concentration Reference
Aspergillus sp.
CNC139
Plinabulin
(diketopiperazine)
Multiple myeloma cells (MM.1S,
MM.1R, RPMI8226,
and INA-6)
2.7 to 3.375 ng/mL (IC50) [42]
Aplidium albicans Plitidepsin (depsipeptide) MCF-7 55.5 ng/mL (IC50) [43]
Halichondria okadai Lurbinectedin (alkaloid)
Ovarian cancer cells (RMG1,
RMG2, KOC7C, HAC2, A2780,
HeyA8 and SKOV-3)
0.78 to 2.34 ng/mL (IC50) [44]
These studies give an overview on the active concentrations for each compound and delimit
a concentration range (0.365–272 ng/mL IC50) that can be used to understand whether a compound
is active enough to be considered as a drug candidate. The sources of the active compounds in all
of these cases were multicellular organisms which implies that there are difficulties in harvesting
biomass and obtaining low amounts of secondary metabolites and a huge environmental impact due
to incorrect harvesting [4]. Most of the compounds in Tables 3 and 4 are small-medium size molecules,
but of particular interest is brentuximab vedotin, an antibody-drug conjugate where the compound
monomethyl auristatin E is linked to a monoclonal antibody (mAb) that recognizes a specific marker
expression in cancer cells and directs monomethyl auristatin E to the targeted cancer cell. The reason
why it is used as a complex and not as a pure compound is because it is too potent (100–1000 times
more potent than doxorubicin, a common drug used for chemotherapy) and less specific to cancer
cells [45].
6. Discussion
Anti-cancer compounds from marine microalgae have been poorly investigated. Most studies
have been done on microalgal extracts or fractions obtained using low resolution methods such
as liquid-liquid partitioning or solid phase extractions. It is uncommon to see dereplication
methodologies, fractionations based on high throughput techniques (e.g., HPLC or gas
chromatography) or a complete structural elucidation of the compounds that have been found.
Despite the low availability of data so far, the studies performed on Chlorella sorokiniana and
Chaetoceros calcitrans show interesting activities compared to commercially available marine anti-cancer
drugs [18,26]. Most of the marine pharmaceuticals on the market are active at the level of 0.6–7 ng/mL
(Table 3) while the fractions from Chlorella sorokiniana and Chaetoceros calcitrans display significant
activity at 1–3 µg/mL (Table 1). Even if fractions and pure compounds cannot be directly compared
in terms of activity, the anti-cancer activities of C. sorokiniana and C. calcitrans extracts seem veryMar. Drugs 2018, 16, 165 14 of 17
promising and appear preferential for further investigation and purification of the active molecules.
Considering that 0.0001% of the crude aqueous ethanol extract of the ascidian Ecteinascidia turbinate [46]
leads to the isolation of trabectedin ET-743 and the development of the anti-cancer drug Yondelis,
it would not be difficult to find a compound from C. sorokiniana and C. calcitrans with an activity as
high as that of the marketed drugs. These data highlight that microalgae can be a promising source
of anti-cancer compounds. Regarding the other fractions/extracts obtained from marine microalgae,
it cannot be excluded that other compounds are present with clinical or biotechnological potential,
but further studies are necessary to demonstrate this possibility.
Several compounds isolated from microalgae mentioned in this review have been studied not
only as possible anti-cancer compounds, but also for other biotechnological applications. Compounds
such as polyunsaturated aldehydes [11,12], eicosapentaenoic acid (EPA) [16], fucoxanthin [17],
violaxanthin [15], stigmasterol [22] or chrysolaminaran [14] (Table 1) may have a potential role as
high-value products (e.g., nutraceuticals, cosmeceuticals, additives, fuel precursors and biomaterials)
or as possible future new drugs. For example, the polyunsaturated aldehydes have shown
anti-proliferative activities on Caco-2 [11], A549 and COLO 205 cancer cell lines [12], but also
anti-bacterial activities [47,48], making them possible new candidates for drug development.
The polyunsaturated fatty acid, EPA, has been widely studied as a nutraceutical or dietary supplement,
with beneficial effects on fetal development, prevention of cardiovascular diseases and even,
the improvement of cognitive functioning in patients with Alzheimer’s disease [49].
The carotenoids, fucoxanthin and violaxanthin, have been used as precursors of vitamins in food
and animal feed, additives, cosmetics, food coloring agents and biomaterials [50]. In particular,
fucoxanthin has been shown to possess potential anti-inflammatory, antioxidant, anti-obesity,
anti-diabetic, anti-tumorigenic and cardioprotective activities [51]. On the market, there are several
fucoxanthin-based products used as dietary supplements such as “Solaray Fucoxanthin Special
Formula Vegetarian Capsules” or “BRI NUTRITION ® Fucoxanthin Capsules”.
Phytosterols such as stigmasterol have been receiving increasing attention because of their capacity
to reduce blood cholesterol concentrations, prevent cardiovascular disorders and because of their health
benefits in general [52]. Stigmasterol has been studied not only for its anti-cancer activity [22] but also
for its antioxidant activity [53]. On the market, the phytosterol, β-sitosterol, is sold as a nutraceutical
under brand names like “NOW ® Foods Beta-Sitosterol Plant Sterols Softgels”. β-sitosterol has been
widely studied for several biological activities such as anti-inflammatory, antioxidant, anti-diabetic or
anti-cancer activities [54].
Finally, microalgal polysaccharides have the capacity to modulate the immune system and
inflammatory reactions, making them interesting candidates for cosmetic additives, food ingredients
and natural therapeutic agents [50]. Chrysolaminaran, for example, is a storage polysaccharide that is
well known to be a common water soluble biopolymer synthetized by diatoms. Water soluble storage
carbohydrates are more accessible, energetically and biophysically, than insoluble starch [30] so they
can also be useful for microalgal energy applications.
There is sufficient evidence confirming the key roles of culturing conditions (e.g., light intensity,
photoperiod, nutrient availability or harvesting time) in the modification of microalgal bioactivity.
Studies such as those by Lauritano et al. [8], Ingebrigten et al. [27] and Ribalet et al. [28] have shown
how light intensity, temperature, nutrient concentration and harvesting time have strong impacts on
the activity of different microalgal strains. The use of stressful conditions (such as nutrient starvation,
light or temperature variation) seems to play a key role in the production of active metabolites.
Unfortunately, very few studies on anti-cancer activities from microalgae have reported the culturing
conditions and the growth phases at which the species were tested in detail (Table 2). This makes
stressful culturing conditions still an unexploited tool with a high potential for the bioprospecting of
novel bioactive metabolites from marine microalgae.
Author Contributions: K.A.M.A., C.L. and A.I. conceived and analyzed the data; K.A.M.A., C.L., A.I. and G.R.
wrote the paper.Mar. Drugs 2018, 16, 165 15 of 17
Funding: This research received no external funding.
Acknowledgments: Kevin A. Martínez Andrade has been supported by a fellowship Marie Skłodowska-Curie
Innovative Training Networks PhD (Project Marpipe MSCA-ITN-ETN Proposal number: 721421). We thank Flora
Palumbo for graphics.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the writing
of the manuscript.
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