103 CHAPTER lll Antitumour activity of the Marine sponge Sigmadocia pumila and Sea cucumber Holothuria atra 3.l. INTRODUCTION Marine invertebrates such as cnidarians, bryozoans, mollusks, tunicates, and echinoderms are well recognized as sources of various natural molecules most often tested against cancer cell lines (Hart et al ., 2000). Rangel et al., (2001) evaluated more than 300 crude extracts obtained from marine sponges, ascidians, echinoderms, bryozoans and octocorals, for cytotoxic activity against colon (HCT8), melanoma (B16) and breast (MCF-7) cancer cell lines. Nakao et al., (2004) reported the isolation of renieramycin-A a new compound from the Japanese sponge Neopetrosia sp. It mainly inhibited recombinant Leishmania amazonensis proliferation, and showed cytotoxicity at ten times higher concentration. Rashid et al., (2002) identified the pellynol- I, a new cytotoxic polyacetylene from the sponge Pellina sp and described pyrinodemins B-D, and Potent cytotoxic bis-pyridine alkaloids from marine sponge Amphimedon sp. Oku et al., (2000) discovered the new isomalabaricane triterpenes from the marine sponge Stelletta globostellata that induced morphological changes in rat fibroblasts and showed significant cytotoxic effects. Qureshi and Faulkner (2007) reported alpha-hydroxytheonellasterol, cytotoxic 4-methylene sterol from the Philippines sponge Theonella swinhoei and then demonstrated the isolation of bioactive 5 alphas, 8 alpha-epidioxy sterols from the marine sponge Luffariella sp. The identification of bioactive compounds from the marine invertebrates using the MeOH/EtOAc (1:1) extracts from the Madagascar sponge, Biemna laboutei was found to be cytotoxic to a series of human tumor cells. From the sponges, seven new guanidine alkaloids, designated as netamines A–G (1–7), have been isolated and their structures were elucidated. Compounds 3 and 4 were found to be cytotoxic against NSCL (A549), colon (HT29), and breast (MDA-MB-231) human cell lines (Hagit et al., 2006). Three new spiculoic acids 1–3 and two
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103
CHAPTER lll
Antitumour activity of the Marine sponge Sigmadocia pumila and Sea
cucumber Holothuria atra
3.l. INTRODUCTION
Marine invertebrates such as cnidarians, bryozoans, mollusks, tunicates, and
echinoderms are well recognized as sources of various natural molecules most often tested against
cancer cell lines (Hart et al ., 2000). Rangel et al., (2001) evaluated more than 300 crude extracts
obtained from marine sponges, ascidians, echinoderms, bryozoans and octocorals, for cytotoxic
activity against colon (HCT8), melanoma (B16) and breast (MCF-7) cancer cell lines. Nakao et
al., (2004) reported the isolation of renieramycin-A a new compound from the Japanese sponge
Neopetrosia sp. It mainly inhibited recombinant Leishmania amazonensis proliferation, and
showed cytotoxicity at ten times higher concentration. Rashid et al., (2002) identified the
pellynol- I, a new cytotoxic polyacetylene from the sponge Pellina sp and described pyrinodemins
B-D, and Potent cytotoxic bis-pyridine alkaloids from marine sponge Amphimedon sp.
Oku et al., (2000) discovered the new isomalabaricane triterpenes from the marine
sponge Stelletta globostellata that induced morphological changes in rat fibroblasts and showed
significant cytotoxic effects. Qureshi and Faulkner (2007) reported alpha-hydroxytheonellasterol,
cytotoxic 4-methylene sterol from the Philippines sponge Theonella swinhoei and then
demonstrated the isolation of bioactive 5 alphas, 8 alpha-epidioxy sterols from the marine sponge
Luffariella sp.
The identification of bioactive compounds from the marine invertebrates using the
MeOH/EtOAc (1:1) extracts from the Madagascar sponge, Biemna laboutei was found to be
cytotoxic to a series of human tumor cells. From the sponges, seven new guanidine alkaloids,
designated as netamines A–G (1–7), have been isolated and their structures were elucidated.
Compounds 3 and 4 were found to be cytotoxic against NSCL (A549), colon (HT29), and breast
(MDA-MB-231) human cell lines (Hagit et al., 2006). Three new spiculoic acids 1–3 and two
104
members of a new closely related family of natural products named zyggomphic acids 4 and 5
were isolated from the marine sponge Plakortis zyggompha. They were of polyketide origin. The
large number of close bioactive analogues showed the preliminary structure–activity relationships
and antitumoural agents (Philippe et al., 2007).
The differential effects of the crude extracts from sponges containing the natural products
may specifically target endocrine growth regulatory pathwasys on MAPK mitogen activated
protein kinase cascade in SW-13 human adrenal carcinoma cells in culture (Ahond et al., 1988).
The MeOH crude extract of the Indonesian marine sponge Xestospongia sp was found to be
effective cytotoxic against the KB cells. The inhibition was due to the presence of the bioactive
compound aaptamine a novel alkaloid (Marielise et al., 2003).
Didemnin B, isolated from a Caribbean tunicate, was a cyclic depsipeptide with potent
antitumor activity and it was the first marine natural product studied clinically. Didemnin B had
inhibited eukaryotic protein synthesis by specifically binding to the GTP-bound EF-1a, an
essential factor for peptide elongation (Schwartsmann et al., 2003). The 13-Deoxytedanolide (13-
DT) was a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens and a
parent compound, tedanolide, isolated from the Caribbean sponge Tedania ignis showed
remarkable cytotoxicity against P388 murine leukemia cells at pico to nano Molar ranges
(Narquizian and Kocienski, 2000). Kijjoa et al., (2001) described the isolation of the
bromotyrosine derivatives fistularin-3, agelorins A and B. The new 11, 17-dideoxyagelorins A
and B halotyrosine derivatives as well as clionasterol a bioactive compound from the marine
sponge Suberea sp in the Gulf of Thailand were identified. It exhibited significant inhibitory
effects against five human cancer cell lines such as MCF-7 (breast), NCI-H460 (lung), SF-268
(CNS), TK-10 (renal) and UACC-62 (melanoma).
Sea cucumbers such as Bohadschia marmorata vitiensis, Stichopus variegatus, Stichopus
badionotus collected from the Malaysian coastal waters (Hawa et al., 1999). They act as the
105
appropriate source of antioxidants for humans. The various activities of sea cucumber extracts
include wound healing promoter, antimicrobial, anticancer and immunomodulatory.
Tong et al., (2005) stated that the potential angiogenesis inhibitors, a novel sulfated
saponin philinopside A, isolated from the sea cucumber Pentacta quandrangulari, possessed dual
antiangiogenic and antitumour effects. It inhibited human microvascular endothelial cells with
minimum inhibitory concentration. Zhang et al., (2006) revealed that fuscocineroside C bioactive
compound obtained from sea cucumber Holothuria fuscocinerea an triterpene glycoside showed
cytotoxic nature against human cancer cells.
Hillaside C a triterpene derived from sea cucumber Holothuria hilla inhibited the
growth of human leukaemia, breast and colon cancer cells in vitro in a dose and time-dependent
manner by a mechanism that required induction of apoptosis and the concomitant reduction of the
apoptosis-suppressing protein Bcl-effect (Wu et al., 2006). Intercedenside D–I isolated a
cytotoxic triterpene glycoside from the sea cucumber Mensamaria intercedens a marine natural
product inhibited proliferation of several human cancer cell lines (Zou et al., 2005). Steroid
glycosides are a class of wide-spread natural products having marine origins. Spirostan and
furostan steroid saponins, pregnane glycosides have a potential to be used as cancer therapies.
Structurally, these glycosides exhibit a moderate cytotoxicity against human leukemia cell lines
(Prassas and Diamandis, 2008). Considering these, the antitumour activity was evaluated in sea
cucumber H. atra as well as in sponge S. pumila.
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3.2. MATERIALS AND METHODS
Cytotoxicity studies
3.2.1. Cell Cultures: Human epidermoid larynx carcinoma cell line (Hep 2) and African green
monkey kidney normal cell line (Vero) were obtained from the King Institute of Preventive
Medicine, Chennai. They were maintained in Dulbecoo’s Modified Eagles medium (DMEM) with
high glucose and glutamine (HiMedia) supplemented with 10% heat inactivated FBS and 1%
penicillin/streptomycin, at 37°C in a humidified atmosphere containing 5.0% CO2.
Cervical cancer cell line (HeLa) and Human breast cancer cell lines (MCF-7) were
obtained from National Centre for Cell Science (NCCS) Pune. Cells of HeLa were grown as
monolayer culture in RPMI-1640 medium with sodium bicarbonate, without L-Gludamine (Hi
media) and incubated at 37°C in a 5% of CO2 incubator whereas the MCF-7 were grown and
maintained using Dulbecoo’s Modified Eagles medium (DMEM).
3.2.2. MTT Assay using HEp2 and Vero cell lines.
This Colorimetric assay was based on the capacity of Mitochondria succinate dehydrogenase
enzymes in living cells to reduce the yellow water soluble substrate 3-(4, 5-dimethyl thiazol-2-yl)-
2, 5-diphenyl tetrazolium bromide (MTT) into an insoluble, colored formazan product which was
measured spectrophotometrically23-24. Since reduction of MTT can only occur in metabolically
active cells, the level of activity is a measure of the viability of the cells. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the
anti-proliferative activities of the S. pumila and H. atra extracts against the HEp2 and Vero cell
lines in twenty four well microtitre plates. Each well was washed with MEM (w/o) FCS twice or
thrice in which 200µl of MTT (5mg/ml) was added and incubated for 6 to 7h in 5% CO2 in
incubator. After incubation, 1.0 ml of DMSO was added in each well, mixed thoroughly by
pipette and left for 45seconds. The presence of viable cells was detected with purple colour due to
the formazan crystals after adding DMSO. The suspension was transferred in to the cuvette of
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spectrophotometer and the O.D values were read at 595nm by keeping DMSO as a blank. Values
were plotted as concentration of the sample on X axis and relative cell viability on Y axis.
3.2.3. MTT assay using Hela cell lines and MCF-7 cancer cell lines.
The cells were preincubated at a concentration of 1 × 106
cells/ml in culture medium for 3
h at 37 °C and 6.5 % CO2. Then, the cells were seeded at a concentration of 5 × 104
cells/well in
100 µl culture medium and at various concentrations of extracts (dissolved in 2% DMSO
dimethylsulphoxide solution) into microplates (tissue culture grade, 96 wells, flat bottom) and
incubated for 24h at 37 °C and 6.5% CO2. The cell proliferation was based on the ability of the
mitochondrial succinate-terazolium reductase system to convert 3-(4,5- dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to a blue colored formazan. The test denoted the surviving
cells after exposure to toxic compounds. Then, 10 µl MTT labelling mixture was added and
incubated for 4h at 37°C and 6.5% CO2. Each experiment was conducted as triplicates sets. Then
100 µl of solubilization solution was added into each well and incubated for overnight. The
spectrophotometric absorbance of the samples was measured using a microplate (ELISA) reader.
The wavelength to measure absorbance of the formazan product in 570 nm according to the filters
available for the ELISA reader was used. The reference wavelength was more than 650 nm.
IC50, the concentration of compound required to inhibit 50 % cell growth, was
determined by plotting a graph of Log (concentration of compound) vs % cell inhibition. A line
drawn from 50 % value on the Y axis meets the curve and interpolates to the X axis. The X axis
value gives the Log (concentration of compound). The antilog of that value gives the IC50 value.
Percentage inhibition of novel compounds against all cell lines was calculated using the following
formula:
(At - Ab)
% cell survival = ------------ × 100
(Ac - Ab)
Where, At = Absorbance of sample (test)
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Ab= Absorbance of blank (Media),
Ac= Absorbance of control (cells)
% cell inhibition = 100 - % cell survival.
3.2.4. Trypan blue dye exclusion test
Being an essential dye, Tryphan blue was used in estimating the number of viable cells
present in a population. The hemacytometer was washed and put on with a cover slip with the
help 70% Isopropanol and blot dried with kim wipe. The hemacytometer with coverslip was
observed under microscope to confirm about their clean lines. The culture sample was mixed to
resuspend cells. 20 µl of cell culture sample was taken and filled into sterile microfuge tube. To
this 20 µl of 0.4% Trypan blue solution was added and mixed well by gently aspirating and
dispensing the solution with the help of micropipette. The coverslip was fixed on the centre top of
the hemacytometer. To the 10 µl mixture of the cell culture the Trypan Blue mixture taken from
the microfuge tube was added and kept in the hemacytometer assembly on microscope stage using
100 X magnification. The live cells were observed as clear and the dead cells as blue in colour.
Beginning with quadrant 1 and moving through to quadrant 4, the cells were counted in a
serpentine fashion. The number of live and dead cells were recorded. The live cell count was
determined as follows:
C = (N/V) x D
Where, C=live cell count in cells per milliliter
N = total number of live cells counted in the four main quadrants
V = volume of counting area. The total volume of the four quadrants is 0.0004mL. (Each quadrant
was 0.0001mL),
D = dilution factor. The percent viability was calculated by using the following formula:
% viability = (live cell count/total cell count) X 100
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3.2.5. Sulphorodamine B assay
Sulphorodamine B (SRB) is a bright pink Aminoxanthine dye with two sulfonic groups.
Under mild acidic conditions, the SRB binds dye to basic amino acid residues in TCA (Trichloro
acetic acid) fixed cells to provide a sensitive index of cellular protein content that is linear over a
cell density range of visible at least two order of magnitude. The monolayer cell culture was
trypsinized and the cell count was adjusted to 0.5 to 1.0 x105
cells/ml using medium containing
10.0% new born sheep serum. To each well of the 96 well microtitre plate, 0.1ml of the diluted
cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was
formed, the supernatant was flicked off, washed once and 100 µl of different test compound
concentrations were added to the cells in microtitre plates. The plates were then incubated at 37oC
for 72 h in 5% CO2 incubator, microscopic examination was carried out, and observations were
recorded every 24 h. After 72 h, 25 µl of 50% trichloroacetic acid was added to the wells gently
such that it formed a thin layer over the test compounds to form overall concentration of 10.0%.
The plates were incubated at 4oC for 1 h.
The plates were flicked and washed five times with tap water to remove traces of
medium, sample and serum and were then air-dried. The air-dried plates were stained with 100 µl
SRB and kept for 30 minutes at room temperature. The unbound dye was removed by rapidly
washing four times with 1% acetic acid. The plates were then air dried. To this 100 µl of 10mM
Tris base was added to solubilise the dye. The plates were shaken vigorously for 5minutes. The
absorbance was measured using microplate reader (Fig 3.14) at a wavelength of 540nm. The
percentage growth inhibition was calculated using the following formula: