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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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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
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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.
106
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
107
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:
Percent cell inhibition= 100-{(At-Ab)/ (Ac-Ab)}x 100
Where,
At= Absorbance value of test compound
Ab= Absorbance value of blank
Ac=Absorbance value of control
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3.2.6. Cytotoxicity studies (In vivo methods)
Animals:
Male Swiss albino mice weighing between 20 to 25g were used for the in vivo cytotoxicity
studies. They were fed with standard pellet diet, water ad libitum and maintained in six cages.
Ethical clearance for the in vivo tumor studies was obtained from the Institutional Animal Ethical
Committee Sankaralingam Bhuvaneswari college of Pharmacy, Sivakasi.
Tumor cell lines:
Daltons ascitic lymphoma (DAL) cells were procured from Amala Cancer Research Institute,
Thrissur, Kerala and transplanted by intra peritoneal inoculation of 1x 106 cells/mouse.
Antitumour activity in mice
After acclimatization, the mature male Swiss albino mice were divided into six groups (n=6).
They were given food and water ad libitum. All the groups except group I were injected with
DAL Cells (1×106
cells/mouse.i.p.). This was taken as day 0. Group I served as normal saline
control (5 ml/kg, p.o.) and Group II served as DAL control. On day 1, the Group lll and lV were
given with the methanol extract of Sigmadocia pumila at a dose of 5 and 10 mg/kg body weight.
Then Group V and Vl were administered orally of Holothuria atra at a dose of 5 and 10 mg/kg
body weight. It was continued for 14 consecutive days. On day 15, three mice of each group were
sacrificed 24 h after the last dose and the rest were kept with food and water ad libitum to check
the increase in the life span of the tumor hosts. The effect of methanolic extract on tumor growth
and host’s survival time were examined by studying the parameters like tumor volume, tumor cell
count, viable tumor cell count, nonviable tumor cell count, mean survival time and increase in life
span.
Determination of tumor volume
The mice were dissected and the ascetic fluid was collected from the peritoneal cavity. The
volume was measured by taking it in a graduated centrifuge tube and packed cell volume was
determined by centrifuging at 1000 g for 5 min (Fig 3.15).
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Determination of tumor cell count
The ascetic fluid was taken in a RBC pipette and diluted 1000 times. Then a drop of the diluted
cell suspension was placed on the Neubauer counting chamber and the number of cells in 64 small
squares was counted.
Estimation of viable tumor cell count
The cells were stained with Trypan blue (0.4% in normal saline) dye. The cells that did not take
up the dye were viable and those that took the stain were nonviable. These viable and nonviable
cells were counted: Cell count = (No. of cells x Dilution) / (Area x Thickness of liquid film)
Percentage increase life span
The mortality was monitored to infer the effect of the S. pumila and H. atra extracts on tumor
growth and the percentage increase in life span (ILS %) was calculated as follows
ILS (%) = [(Mean survival of treated group/ Mean survival of control group)-1] x100
Mean survival time = [1st Death + Last Death] / 2
Hematological studies
The effect of Sigmadocia pumila and Holothuria atra extracts on peripheral blood was
investigated. The RBC, WBC counts and estimation of hemoglobin were done by standard
procedures from freely flowing tail vein blood. Packed cell volume (PCV) was determined by the
method described by Armour et al., (1965).
Statistical analysis
The experimental results were expressed as the mean ± S.D. Data were assessed by the method of
One way ANOVA followed by Dunnett post hoc test (Prasad and Giri, 1994). P value of <0.01
was considered as statistically significant.
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3.3. RESULTS
The present investigation was carried out to evaluate the antitumour activity of
Sigmadocia pumila and Holothuria atra using in vitro and in vivo methods. The Table 3.1.
denotes the cell lines used in the study. The cell lines were of epithelial cells. They include the
(Vero) an animal cell line, (Hep2) human larynx carcinoma cell line, (Hela) cervical cancer cell
line and MCF-7 Breast cancer cell line.
From the MTT assay using 24 well plates, it could be noted that the sea cucumber extracts
reduced the viability of the Hep2 and Vero cell lines (Table 3.2 and Table 3.3). The concentration
of the extract used for this assay ranged from 0.078 to 10 mg/ml and at the lower concentrations,
the survival of the cells was high whereas at higher concentrations the survival rate was less. It is
possible that cell death induction could be attributed to the presence of bioactive compounds. The
cell inhibition was also seen in Vero cell lines and when compared to Hep2 cell lines, the effect
was less. Thus, the extracts of H. atra possessed antitumor activity and it could be referred from
Fig 3.1 and Fig 3.2. The graphs represented the H. atra extract against the Hep2 and Vero cell
lines. The concentration of the extracts ranged from 0.078mg/ml to 10mg/ml and were plotted
against the inhibition of cells.
The monolayer of Hep2 cell lines and the cytotoxic effects of the extracts in Hep2 cell
lines were as 25, 50, 75 and 100% dead cells (Fig3.3). Similarly, the monolayer of Vero cell lines
and the cytotoxic effects of extracts in the Vero cell lines for 25, 50, 75, 100% dead cells using
inverted microscope are presented in Fig 3.4. In Table 3.4 and Table 3.5, the details of cytotoxic
effect of the sponge Sigmadocia pumila against the action of Hep2 and Vero cell lines using MTT
assay at 595nm are presented. Increase in concentration of the extracts showed tremendous effect
of the cell inhibition. Absorbance values at lower levels indicated the reduction in the rate of cell
proliferation. Higher absorbance caused apoptosis against the Hep2 and Vero cell lines. The
concentrations of the extracts ranged from 0.078mg/ml to 10mg/ml were plotted against the
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inhibition of cells. The results of the MTT assay using the crude extracts of Sigmadocia pumila
against the Hep2 and Vero cell lines are graphically represented in Fig 3.5 and Fig 3.6. It was
found that increased concentration of the extract with less absorbance values caused more death
of the cells. An increase in cell viability indicated cell growth, while a decrease in viability could
be interpreted as the result of toxic effects of the extract.
The efficacy for screening of S. pumila extracts on Hep2 and Vero cell lines to detect the
cytostatic potential of anticancer activity can be assessed by means of quantifying cell growth.
The cytotoxic effects in images of Hep2 and Vero cell lines are observed through an inverted
microscope. The Fig 3.7 depicts the cytotoxic levels of the sponge S. pumila against the Hep2 cell
lines at 25, 50, 75 and 100% dead cells. Fig 3.8 shows the effect of S. pumila against Vero cell
lines.
From Table 3.6 and Table 3.7, the amount of the extract of sponge S. pumila and sea
cucumber H. atra required to inhibit 50% of the antitumour activity against the Hela cell lines in
96 well plates could be determined. Antitumor activity was measured by using the IC50 values.
The antiproliferative effect (IC50 value) of the Sigmadocia pumila was 265.0 against the cervical
cancer cell line (Hela), whereas the IC50 value exhibited by the Holothuria atra was 468.0 against
the cervical cancer cell line (Hela).
The graphical data for the antitumour effect of the Sigmadocia pumila and Holothuria atra
are given in Fig. 3.9 and 3.10. The cell inhibition was determined from the extract concentration
ranged from 0.078mg/ml to 10mg/ml. The absorbance values were measured at 570nm.
Percentage of growth inhibition was identified at different concentration of the extracts. The
gradual decrease in absorbance values showed increase in inhibition effect of the crude extracts
against the Hela cell lines.
The results of antitumour activity of the crude methanolic extracts of S. pumila and H. atra
against the breast cancer cell lines MCF-7 in 96 well microtitre plates are given in Table 3.8 and
114
Table 3.9. The data revealed a significant inhibition of extracts ranges from 0.078mg/ml to
10mg/ml. It could be observed that the extracts have influenced considerable activity over the
MCF-7 cell lines. The susceptibility of cells to the extract exposure was characterized by IC50
values. Results indicated that the antiproliferative effect increased with the increase in
concentration of the extracts. The IC50 value for the sponge Singmadocia pumila was 432.0. The
IC50 value for the sea cucumber Holothuria atra was 352.0
Fig 3.11 and Fig 3.12 provide the dose response curves constructed between the range
of 0.078mg/ml and 10mg/ml for the test extracts from Sigmadocia pumila and Holothuria atra
against the breast cancer cell lines MCF-7. A decrease in number of viable cells with the increase
in concentration of the extracts was noted (Table 3.10 and Table 3.11) Results of cell counting
and viability of cells using tryphan blue staining were indicators for the influence of extracts.
The sponge Sigmadocia pumila extracts showed an inhibition of 87.27% against the
Hep2 cell line with the 1.92 x 105 dead cells in the total cell count of 2.20 x 10
5 at the pH 7.5. Cell
inhibition of 79.0 % was noted against the Vero cell line with the 1.85 x 105 dead cells in the total
cell count of 2.34 x 105
at the pH7.5. For the Hela cell line, the cell inhibition was 70.83 % with
1.70 x 105 dead cells in the total cell count of 2.40 x 10
5 at the pH 6.9 The cell inhibition of 87.50
% was noted against the MCF-7 cell line with 1.75 x 105 dead cells in the total cell count of 2.0 x
105
at the pH 7.2 (Table 3.10).
The sea cucumber Holothuria atra extracts gave a cell inhibition of 87.82% against the
Hep2 cell line with the 2.02 x 105 dead cells in the total cell count of 2.30 x 10
5 at the pH 7.5
(Table 3.11). It stated the cell inhibition of 81.13% against the Vero cell line with the 1.72 x 105
dead cells in the total cell count of 2.12 x 105
at the pH 7.5. Further, it was analysed that the cell
inhibition of 81.81% against the Hela cell line with the 1.80 x 105 dead cells in the total cell
count of 2.20 x 105
at the pH 6.9. Finally the cell inhibition of 72.72% against the MCF-7 cell
line with the 1.76 x 105 dead cells in the total cell count of 2.42 x 10
5 at the pH 7.5. The cell
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proliferation and inhibition measurment of the sea cucumber Holothuria atra show that it can be
developed as an antitumour agent. These observations of Hep2, Vero, Hela and MCF-7 cell lines
using Sigmadocia pumila and Holothuria atra extracts using tryphan blue staining are represented
in Table 3.10 and Table 3.11.
The results of Sulphorodamine B (SRB) assay which explain the antitumour effects of
the extracts of the S. pumila and H. atra at 540nm are in Table 3.12 and Table 3.13. The SRB
assay showed significant effect against the Hep2 and HeLa cell lines of S. pumila with the growth
inhibition of cells at 90% and 85%. It was found that MCF-7 and Vero cell lines had inhibitory
activity of S. pumila extracts at 82% and 75%.
Holothuria atra extracts were screened for determining its potential cytotoxicity against
HeLa and Hep2 cell lines which displayed an inhibition level of 88% and 85%. From the MCF-7
and Vero cell line at 78 and 60% considerable activity was seen with the growth inhibition of
cells. Fig 3.13 and Fig 3.14 provide the graphical representation and comparative view of the
extracts of Sigmadocia pumila and Holothuria atra against the Hep2, Vero, Hela and MCF-7 cell
lines. The percentage growth inhibition increased with an increasing concentration of extract
steadily up to 0.0196mg/ml. The antitumour effects of the sponge Sigmadocia pumila and sea
cucumber Holothuria atra using Hela and MCF-7 cell lines could be noted from Fig 3.15.
Absorbance values that were lower than the control cells indicated a reduction in the rate
of cell proliferation. Conversely, a higher absorbance rate indicated an increase in cell
proliferation. This led to cell death. It can be seen by morphological changes. Since reduction of
MTT can only occur in metabolically active cells, the level of activity is considered as a reliable
measure of the viability of the cells. Table 3.14 indicates the evaluation of antitumor effects of the
sponge S. pumila and H. atra crude extracts in DAL tumor bearing mice through in vivo approach.
The values were analysed using statistical analysis by means of standard deviation and one way
ANOVA. The mice were divided into six groups each having six individuals and maintained in
116
separate cages. The doses 5mg/kg and 10mg/kg were selected. The results from Table 3.14
showed the comparative values of Sigmadocia pumila extract treated and DAL control animals.
Tumor volume, packed cell volume and viable cell count decreased significantly (p < 0.01) with
the administration of increase in dose of Sigmadocia pumila extract at 5 and 10 mg/kg
respectively.
Non-viable cell count was significantly (p <0.01) higher in Sigmadocia pumila extract
treated animals when compared with DAL control animals. A regular rapid increase in ascetic
tumor volume was observed in DAL tumor bearing mice. Ascetic fluid is the direct nutritional
source for tumor cells and a rapid increase in ascetic fluid with tumor growth would be a means to
meet the nutritional requirement of tumor cells (Fig 3.16). Furthermore, the median survival time
was increased to 42.8±2.5 (%ILS = 51.3) and 48.3±2.2 (%ILS = 75.08) on administration of S.
pumila extract at 5 and 10 mg/kg dose respectively. All these results clearly indicated that the S.
pumila had a remarkable capacity to inhibit the growth of solid tumor induced by DAL cell line in
a dose-dependent manner in experimental animals. The reliable criteria for judging the value of
any anticancer drug is the prolongation of the life span of animals. It may be concluded that
Sigmadocia pumila by decreasing the nutritional fluid volume and arresting the tumor growth
increases the life span of DAL bearing mice.
The hematological parameters were analysed in the mice from group 1 to 6 and the values
are given in Table 3.15. The values are represented by statistical analysis by calculating the
standard deviation and One way ANOVA. It is evident that on 14th day the tumor bearing mice
were found altered significantly when compared to the normal group in terms of hematological
parameters. In the DAL control set the total WBC count increased and there was a significant
reduction in Hb content. In percentage of differential count the neutrophil was increased where as
the lymphocyte count decreased. The administration of S. pumila extract with both 5 and 10mg/kg
doses altered the depleted parameters towards near normal. The higher dose (10mg/kg) of
117
Sigmadocia pumila was more effective. Thus, the S. pumila extract exerted antitumor activity
against DAL bearing mice.
The effect of Holothuria atra on the survival of tumour bearing mice showed that the
DAL treated animals were found to be having the tumor volume of 4.9±0.94. Holothuria atra
extract treatment at both dose level significantly (p < 0.05) reduced tumour volume which was
found to be 2.40±0.11 and 1.01±0.04 respectively. Viable cell count of the tumor bearing mice
was significantly decreased while non- viable cell count increased in Holothuria atra extracts
treated groups in dose dependant fashion when compared with DAL treated group.
Moreover, hematological parameters for Holothuria atra tumour bearing mice on day 15
were found to be significantly altered from normal group (Table 3.15). The total WBC count, and
PCV were found to be increased with a reduction of the haemoglobin and RBC. In a differential
count of WBC, the percent of neutrophils increased while the lymphocyte count decreased. At the
same time interval, H. atra extract treatment could change those altered parameters to near
normal.
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Table 3.1: Details of cell lines used in the study
Cell line
Morphology
Origin Species Ploidy Characteristics Supplier
Hep 2 Epithelial Larynx Human Polyploid
Plating
efficiency
high
Kings Institute,
Chennai.
Vero Epithelial Kidney Monkey Aneuploid Viral substract
and assay
Kings Institute,
Chennai
HeLa Epithelial Cervix Human Aneuploid G6PD type A NCCS, Pune
MCF-7 Luminal
epthelial Breast Human Aneuploid
Transfection
host NCCS, Pune
Table 3.2.Cytotoxicity of Holothuria atra on Hep2 Cells by MTT assay
Fig 3.1. Graphical analysis for Holothuria atra on Hep2 cell lines
Concentration
(mg/ml)
Dilution Absorbance
(595nm)
Percentage of
cell inhibition
0.078 1:64 0.44 97.77
0.156 1:32 0.43 97.55
0.3125 1:16 0.42 93.33
0.625 1:8 0.38 84.44
1.25 1:4 0.34 75.55
2.5 1:2 0.31 68.88
5 1:1 0.29 64.44
10 Neat 0.14 31.11
Cell control - 0.45 100
Cell rounding Cell shrinkage Cell death
0
20
40
60
80
100
120
0.0
78
0.1
56
0.3
12
5
0.6
25
1.2
5
2.5 5
10%
Cel
l in
hib
itio
n
Concentration of extracts
(mg/ml)
119
Table 3.3.Cytotoxicity of Holothuria atra on Vero Cells by MTT assay
Fig 3.2. Graphical analysis for Holothuria atra on Vero cell lines
0
10
20
30
40
50
60
70
80
%C
ell
inh
ibit
ion
n
Concentration
(mg/ml)
Dilution Absorbance
595nm
Percentage of
cell inhibition
0.078 1:64 0.66 91.66
0.156 1:32 0.52 72.22
0.3125 1:16 0.44 61.11
0.625 1:8 0.38 52.77
1.25 1:4 0.30 41.66
2.5 1:2 0.22 30.55
5 1:1 0.19 26.38
10 Neat 0.14 19.44
Cell control - 0.45 100
Cell rounding Cell shrinkage Cell death
Concentration of extracts
(mg/ml)
120
Fig 3.3. Cytotoxicity of Holothuria atra extract on Hep2 cell lines
Normal HEP 2 cell line 1+Cytotoxicity effect
2+Cytotoxicity effect 3+Cytotoxicity effect
4+Cytotoxicity effect
1+ For 25% dead cells
2+ For 50% dead cells
3+ For 75% dead cells
4+ For 100% dead cells
121
Fig.3.4. Cytotoxicity of Holothuria atra extract on Vero cell lines
Toxicity 1 Toxicity 2
Toxicity 3 Normal Vero cell line
122
Table 3.4.Cytotoxicity of Sigmadocia pumila on Hep2 Cells by MTT assay
Fig 3.5. Graphical analysis for Sigmadocia pumila on Hep 2 cell lines
0
50
100
150
0.0
78
0.1
56
0.3
12
5
0.6
25
1.2
5
2.5 5
10%
Cel
l in
hib
itio
n
Concentration
(mg/ml)
Dilution Absorbance
(595nm)
Percentage
cell inhibition
0.078 1:64 0.51 98.32
0.156 1:32 0.43 97.85
0.3125 1:16 0.42 97.20
0.625 1:8 0.36 85.00
1.25 1:4 0.32 72.64
2.5 1:2 0.30 65.38
5 1:1 0.28 60.30
10 Neat 0.20 32.00
Cell control - 0.45 100
Cell rounding Cell shrinkage Cell death
Concentration of extracts
(mg/ml)
123
Table 3.5.Cytotoxicity of Sigmadocia pumila on Vero Cells by MTT assay
Fig 3.6. Graphical analysis for Sigmadocia pumila on Vero cell lines
0
20
40
60
80
100
%C
ell
inh
ibit
ion
Concentration
(mg/ml)
Dilution Absorbance
(595nm)
Percentage
cell inhibition
0.078 1:64 0.72 90.10
0.156 1:32 0.68 81.20
0.3125 1:16 0.62 72.00
0.625 1:8 0.56 64.40
1.25 1:4 0.48 50.32
2.5 1:2 0.32 42.60
5 1:1 0.26 30.50
10 Neat 0.18 26.30
Cell control - 0.45 100
Cell rounding Cell shrinkage Cell death
Concentration of extracts
(mg/ml)
124
Fig 3.7. Cytotoxicity of Sigmadocia pumila extract on Hep 2 cell lines
Toxicity 1 Toxicity 2
Toxicity 3 Hep 2 cell control
125
Fig 3.8. Cytotoxicity of Sigmadocia pumila extract on Vero cell lines
3+cytotoxicity 4+cytotoxicity
2 +cytotoxicity 1+cytotoxicity
Normal Vero cell line
1+ For 25% dead cells
2+ For 50% dead cells
3+ For 75% dead cells
4+ For 100% dead cells
126
Table 3.6. Determination of cytotoxicity for Sigmadocia pumila by MTT assay using Hela cell
lines
Fig 3.9. Graphical analysis for Sigmadocia pumila on Hela cell lines
0
20
40
60
80
0.0
78
0.1
56
0.3
12
5
0.6
25
1.2
5
2.5 5
10
%ce
ll i
nh
ibit
ion
Concentration
(mg/ml)
Absorbance
(570nm)
% inhibition IC50
0.078 1.91 42.66
265.0
0.156 1.74 51.29
0.3125 1.73 51.79
0.625 1.65 55.62
1.25 1.63 57.04
2.5 1.62 57.54
5 1.56 60.56
10 1.51 62.61
Cell control 0.45 100
Concentration of extracts
(mg/ml)
127
Table 3.7. Determination of cytotoxicity for Holothuria atra by MTT assay using Hela cell lines
Fig 3.10. Graphical analysis for Holothuria atra on Hela cell lines
0
10
20
30
40
50
60
70
80
90
100
%C
ell
inhib
itio
n
Concentration
(mg/ml)
Absorbance
(570nm)
% inhibition IC50
0.078 0.125 40.56
468.0
0.156 0.103 52.12
0.3125 0.10 60.42
0.625 0.093 68.30
1.25 0.085 75.34
2.5 0.070 83.26
5 0.069 88.32
10 0.032 90.56
Cell control 0.45 100
Concentration of extracts
(mg/ml)
128
Table 3.8. Determination of cytotoxicity for Sigmadocia pumila by MTT assay using MCF-7 cell
lines
Fig 3.11. Graphical analysis for Sigmadocia pumila on MCF-7 cell lines
0
10
20
30
40
50
60
70
80
90
100
%C
ell
inh
ibit
ion
Concentration
(mg/ml)
Absorbance
(570nm)
% inhibition IC50
0.078 1.38 28.42
432.0
0.156 1.08 36.75
0.3125 0.80 45.32
0.625 0.72 54.60
1.25 0.65 66.52
2.5 0.50 75.43
5 0.43 83.62
10 0.32 90.53
Cell control 0.45 100
Concentration of extracts
(mg/ml)
129
Table 3.9. Determination of cytotoxicity for Holothuria atra by MTT assay using MCF-7 cell
lines
Fig 3.12. Graphical analysis for Holothuria atra on MCF-7 cell lines
0
10
20
30
40
50
60
70
80
%C
ell
inh
ibit
ion
Concentration
(mg/ml)
Absorbance
(570nm)
% inhibition IC50
0.078 0.120 30.22
352.0
0.156 0.102 35.64
0.3125 0.10 48.72
0.625 0.088 55.60
1.25 0.085 60.62
2.5 0.075 65.20
5 0.062 70.22
10 0.052 75.60
Cell control 0.45 100
Concentration of extracts
(mg/ml)
130
Table 3.10.Percentage cell inhibition and characterization of cell line exposed to Sigmadocia
pumila using tryphan blue
Cell line % cell inhibition Dead cell count Total cell count pH
Hep2 87.27 1.92 x 105
2.20 x 105
7.5
Vero 79.0 1.85 x 105
2.34 x 105
7.5
HeLa 70.83 1.70 x 105
2.40 x 105
6.9
MCF-7 87.5 1.75 x 105
2.0 x 105
7.2
Table 3.11.Percentage cell inhibition and characterization of cell line exposed to Holothuria atra
using tryphan blue
Cell line % cell inhibition Dead cell count Total cell count pH
Hep2 87.82 2.02 x 105
2.30 x 105
7.5
Vero 81.13 1.72 x 105
2.12 x 105
7.5
HeLa 81.81 1.80 x 105
2.20 x 105
6.9
MCF-7 72.72 1.76 x 105
2.42 x 105
7.2
131
Table 3.12.Determination of Cytotoxicity for Sigmadocia pumila by Sulphorodamine B assay.
Cell lines control
% Growth inhibition
of cells using
Sigmadocia pumila
Hep2 82.20 90.25
Vero 64.52 75.60
Hela 72.46 85.20
MCF-7 74.65 82.42
Cell lines
Fig.3.13. Growth inhibition of cells in SRB assay using the methanolic extracts of Sigmadocia
pumila
0
10
20
30
40
50
60
70
80
90
100
Hep2 Vero HeLa MCF-7
Cell line
Control
Per
cen
tag
e ce
ll i
nh
ibit
ion
132
Table 3.13. Determination of Cytotoxicity for Holothuria atra by Sulphorodamine B assay.
Cell lines control
% Growth inhibition
of cells using
Holothuria atra
Hep2 81.10 85.45
Vero 52.32 60.50
Hela 76.55 88.70
MCF-7 70.22 78.62
Cell lines
Fig.3.14. Growth inhibition of cells in SRB assay using the methanolic extracts of Holothuria
atra
0
10
20
30
40
50
60
70
80
90
100
Hep2 Vero HeLa MCF-7
Cell line
Control
Per
cen
tag
e ce
ll i
nh
ibit
ion
133
Table 3.14. Effect of methanolic extract of Sigmadocia pumila and Holothuria atra on Tumor
volume, tumor cell count, viable tumor cell count, nonviable tumor cell count, mean survival time
and increase in life span in DAL bearing mice (In vivo analysis).
Treatment group
(N=6)
Tumor
volume
(ml)
Packed
cell
volume
(ml)
Tumor cell count
Median
survival
time (days)
%
increas
ed life
span Viable 1 x
106cells/mo
use
Non viable 1
x 106Cells/
mouse
Normal saline
(5ml/kg p.o)
Group 1
- - - - - -
DAL control
(1 x 106 cells)
Group ll
4.9±0.94 3.5±0.12 10.6±0.31
**
0.4±0.03 23.2±1.31** -
Sigmadocia
pumila
(5mg/kg p.o)
+ DAL
Group lll
3.2±0.93
**
2.0±0.14
**
5.6±0.05
**
0.6±0.03 42.8±2.5** 51.3
Sigmadocia
pumila
(10mg/kg p.o)
+ DAL
Group 1V
2.2±0.53
**
0.9±0.21
**
3.2±0.07
**
0.8±0.04 48.3±2.2** 75.08
Holothuria atra
(5mg/kg p.o)
+ DAL
Group V
2.40±0.11
**
1.6±0.10
**
3.89±0.04
**
1.91±0.09 30.8±1.02** 43.50
Holothuria atra
(10mg/kg p.o)
+ DAL
Group Vl
1.01±0.04
**
0.8±0.13
**
2.62±0.09
**
2.14±0.19 35.64±1.05
**
66.32
Statistically significance (p) calculated by one way ANOVA followed by Dunnetts test.
** P < 0.01 calculated by comparing treated groups with DAL control group
134
Table 3.15. Effect of methanolic extract of Sigmadocia pumila and Holothuria atra on
hematological parameters
Treatment group
Hb
content
RBC
cells/ml
X 106
WBC
cells/ml
X 106
Percentage differential count
Lymphocytes Neutrophils Monocytes
Normal saline
(5ml/kg p.o)
16.3±0.2
**
6.1± 0.8
7.9±0.4
69±1.3
28±4.2 2.2±0.1
DAL control
(1 x 106
cells)
8.2±1.1
**
2.9±0.14 16.4± 0.12 21 ±.07 78.1.7 3.8±0.5
Sigmadocia
pumila
(5mg/kg p.o)
+ DAL
14.8±1.3
**
4.6±0.06
11.2±0.21 55+1.2 43.1.44 *1.6±0.3
Sigmadocia
pumila
(10mg/kg p.o)
+ DAL
15.6±0.12
**
5.1±0.2
**
9.8±0.08 57±3.8 41±2.2 *1.7±0.12
Holothuria atra
(5mg/kg p.o)
+ DAL
11.2±0.6
**
5.1±0.5
**
8.7±0.22
55.8± 1.1
30.1±2.2
2.11 ±0.3
**
Holothuria atra
(10mg/kg p.o)
+ DAL
14.4±0.4
**
6.3±0.11
**
11.2±.7 62.3± 2.1 28.12 2.8 6 ±0.3
**
Statistically significance (p) calculated by one way ANOVA followed by Dunnetts test.
* P < 0.05, ** P < 0.01, calculated by comparing treated groups with DAL control group
135
a) Sigmadocia pumila b) Holothuria atra (Hela cell lines)
a) Sigmadocia pumila b) Holothuria atra (MCF-7 cell lines)
Fig 3.15 Cytotoxicity images of Hela and MCF-7 cell lines observed in inverted microscope
136
(a)
(b)
Fig 3.14 (a and b) Sulphorodamine B assay using 96 well microtitre plates of sponge
a)Sigmadocia pumila and Sea cucumber b) Holothuria atra
137
Fig 3.16. Collection of ascetic fluid from the peritoneal cavity of tumor induced mice after
dissection
138
3.4. DISCUSSION
Recent studies in the field of cancer research have revealed promising compounds,
isolated from marine derived natural sources, with proven anticancer activity. Three examples are
trabectedin, cytarabine and eribulin mesylate and they are manufactured under the trade names
such as Yondelis at PharmaMar; Cytosar-U at , Bedford, Enzon, Halaven at Eisai Inc (Gradishar,
2011). The antitumour activity of the marine sponge Sigmadocia pumila and sea cucumber
Holothuria atra methanolic extracts using in vitro and in vivo methods was evaluated. S. pumila
and H. atra showed a high degree of antitumour activity against the Human epidermoid larynx
carcinoma cell line (Hep 2), African green monkey kidney normal cell line (Vero), Human
cervical cancer cell line (HeLa), Human breast cancer cell lines (MCF-7) and Daltons ascites
lymphoma (DAL) cell lines of in vitro and in vivo studies.
MTT assay
In MTT assay, Sigmadocia pumila extracts displayed potent cytotoxic effects against
the Vero, Hep2, Hela and MCF-7 cell lines. The quantification of cellular growth, including
proliferation and viability was detected in this assay using tryphan blue as indicator in this
method. The antitumour activity was measured by identifying the cell viability of extracts and
absorbance at 595nm. Laura et al., (2009) investigated a new cyclic diamine, 1,5-
diazacyclohenicosane, isolated from the marine sponge Mycale sp using methanolic extract which
showed cytotoxic activity against three human tumour cell lines, including lung (A549), colon
(HT29) and breast cancer cells (MDA-MB-231). Wright et al., (2007) extended the current
knowledge on the mechanism of Neopeltolide derived from Neopeltidae sponge a potent inhibitor
of human cancer cell lines, which was active against A-549 and NCI-ADR-RES, and the P388
murine leukemia cell lines. Similarly sponge S. pumila had potent compounds such as cyclic
peptides and lipoglycodepsipeptides. These compounds could induce cell death by apoptosis and
act as inhibitors against the Hep2 cells. The cell inhibition was maximal (98%) in Hep2 cell lines.
139
The antiproliferative activity showed a dose-dependent manner in the marine sponge
Cinachyrella apion which was noted for bioactive molecules. The antiproliferative activity of
lactose binding lectin against Hela, PC3 and 3T3 was seen. In all the assays growth inhibition was
noted in a dose dependent way and as per the increase in concentration (Rabelo et al., 2012).
Simillar effect was seen in Sigmadocia pumila extracts against the cervical adenocarcinoma (Hela
cells) at the increased concentarion of .078mg to10 mg/ml extracts. It was presumed that S.
pumila extracts could have the possibility to control the growth of tumour cells by reducing the
cell growth, division, differentiation and cause death (via apoptosis) which was comparable to that
of mitogen-activated protein kinase (MAPK) pathways.
The antitumour activities seen in the extracts of S. pumila could be attributed to the
availability of effective compounds like 1,2 Benzisoxazole, rotoxamine, and isobenzofuran.
Hence these could act as mediators of cell cycle and inhibited the growth of tumour cells. The
puupehenol segments, isolated from the marine sponge Dysidea sp., along with the known
metabolites, puupehenone and bispuupehenone displayed a wide range of biological properties
such as angiogenesis inhibitors and antitumor activities (Utkina et al., 2011). The morphological
changes and changes in membrane permeability were easily inferred from the images observed
through the inverted microscope among the cell lines of Vero and Hep2. These results suggested
the cytotoxicity activity of the extracts leading to the death of tumour cells.
Detailed analysis from the Fijian sponges of the genus Zyzzya yielded nine
pyrroloiminoquinones of the makaluvamine, batzelline, and isobatzelline/damirone classes.
Comparative testing of these compounds in the National Cancer Institute against 60 tumour cell
lines such as A549, HOP92, SF-539, SNB-19, LOX, M-14 and MCF-7 showed potent
cytotoxicity in MTT assay. Makaluvamine H emerged as the most potent compound isolated from
the sponge Latrunculia purpurea (Genevieve et al., 2005). In the case of S. pumila, presence of
Benzamdines and isobenzofuran which could act as an antiproliferative agent against the MCF-7
140
and Vero cell lines were recorded in this present study. It showed the highest growth inhibition
and reduced cell growth of 87% and 70% at a dose-dependent manner respectively.
Patrizia et al. (2007) stated that a series of 10 bis-indolylpyrazoles of type 9, 10 obtained
by cyclization of diketones using hydrazine monohydrate. These were evaluated against tumour
cell lines such as the colon cancer, ovarian cancer, myeloma which showed antiproliferative
activity. The results obtained measured by TGI and LC50 values had positive response on 91%
and 41% of the tested cell lines, respectively. In S. pumila the half maximal inhibitory
concentration (IC50) of Hela and MCF-7 cell lines was 265 and 432 respectively in MTT assay
indicating the possibility that the extracts were able to interfere with cell cycle arrest in cell lines.
The extracts of S. pumila inhibited the growth of tumour cells such as Hep2, Hela and
MCF-7 possibly due to their positive influence in controlling the growth of tumour cell
proliferation and differentiation. The S. pumila could also stop the mechanism of progressive
transformation of normal human cells to highly malignant derivatives. Park et al., (2006)
described the suppression of U937 human monocytic leukemia cell growth by dideoxypetrosynol
A, a polyacetylene from the sponge Petrosia sp., via induction of Cdk inhibitor p16 and
downregulation of pRb phosphorylation. Peloruside A, a compound isolated from the marine
sponge Mycalehentscheli reduced growth and enhanced apoptosis in H-rastransformed cells.
Interestingly, it showed that the compound was more cytotoxic in oncogene-transformed cells,
which were subsequently blocked in G2/M phase of the cell cycle, leading to apoptosis
independent of caspase activation (Miller et al., 2004). The cytotoxic effects of S. pumila could be
due to either apoptosis inducement or non-apoptotic cell death stimulation. The different
mechanisms of action, like stimulation of autophagic cell death, decrease in NO production in
cells, or cytoskeleton integrity disassembly could determine the antitumour activity of the extracts
and thus requires further investigations.
In the case of H. atra, compounds such as ligustilides, isoquinoline alkaloids and
polysaccharides seen in them could have acted as inhibitors in regulating the metabolism and
141
growth of tumour cells. The findings suggest that H. atra showed cell inhibition to the tune of
90% to the maximum in Hela cells and 75% of cell inhibition in MCF-7 cells. Five cerebrosides,
PA-0-1, PA-0-5, PA-2-5, PA-2-6 and CE-2c were reported from the Japanese sea cucumber
Pentacta australis (Higuchi et al., 1994b). A ganglioside molecular species SJG-1, isolated from
the sea cucumber Stichopus japonicus. SJG-1 possessed a sialic acid, nonhydroxy fatty acids and
phytosphingosine-type long chain bases as major ceramide components. SJG-1 exhibited
neuritogenic activity towards the rat pheochromocytoma cell line PC12 cells in the presence of
nerve growth factor (Kaneko et al., 1999). Additionally, a cerebroside isolated from the sea
cucumber Stichopus japonicas showed effective antitumour activity (Hayashi et al., 1990).
Transcaryophyllene a sesquiterpene and tetrahydrodeoxycorticosterone seen in
Holothuria atra could be considered as antitumour compounds exhibiting cytotoxic effects. From
the MTT assay maximum antitumour activities were observed in Hep2, Hela and MCF-7 cell lines
and little less effect was seen on Vero cell lines. Previous researches on cytotoxic properties of
echinoderm extracts were mostly focused on their glycosides, particularly the saponin and
saponin-like components. Cytotoxic activities were reported on active components like the
propanol extracts from H. impatiens. Glycosides such as the hemoiedemosides from Hemoiedema
spectabilis and a lectin from brown sea cucumbers were detected with antitumour activities (Gana
and Merca, 2002).
Promising in vitro cytotoxic compounds such as the Calcigeroside B, C1 and C2
identified from the holothurians included triterpene glycosides. They showed antiproliferative
action against the human and murine tumour cell lines (Alejandro and Gustafson, 2003). In the
present study, the tumour growth was inhibited by Holothuria atra extract and dosage was an
important criteria which influenced the efficacy of the cell death in Hep2, Hela, Vero and MCF-7
cell lines. The variations in the activity among the sponge and holothuria were determined by the
IC50 of each extract against the particular cell line. These suggest the possibility of apoptotic cell
death through the activation of Bax a proapoptotic protein.
142
Presence of compounds in Holothuria atra such as phenylpropanolamine,
phendimetrazine and ethosuxinide and various hydrophobic compounds could induce the
antiproliferative effects. In Holothuria atra the IC50 values measured against the Hela and MCF-7
were 468 and 352. It exhibited antiproliferative effects. The triterpenoids isolated from sea
cucumbers and other components in sea cucumbers which showed high cytotoxic activities
included echinosides, holothurins, holotoxin and cucumarioside. The inhibitory concentrations
(IC50) detected in sea cucumber Stichopus chloronotus extracts against A549 and C33A cells were
measured as 220 and 382 (Popov, 2002).
Information on the antitumour activity of H. atra has not been reported. Compounds
such as propylhexadrine, phenypropanolamine and transcaryophyllene seen in H. atra could be an
inhibitor for the progression of tumour cells such as Hep2, MCF-7 and Hela. The strong growth
inhibitory activity found in H. atra organic extracts has the ability to inhibit the progression of
various human tumor cells such as Hep2 at 87%, Hela at 81% and MCF-7 cell lines at 72%
respectively. It was detected by tryphan blue exclusion method. However new sphingosine type
cerebrosides, such as CE-2b, CE-2c, and CE-2d were isolated from the Japanese sea cucumber
Cucumaria echinata which showed antitumour activity (Higuchi et al., 1994a). Similarly two
gangliosides obtained from the holothurians Cucumaria japonica exhibited the cytotoxic effect
against the human tumour cell lines (Chekareva et al., 1991).
Angiogenesis inhibitors and aromatase inhibitors present in sea cucumbers play a major
role in reducing the growth of breast cancer and prostate cancers, especially the solid tumors.
Research results showed that angiogenesis inhibitors effectively block the growth of tumours by
cutting off their nutrient and blood supply. The mechanism by which they block tumor growth is
driven by the inhibition of receptor tyrosine kinases (RTKs) that are overexpressed by cancer cells
(Chi, 2006). Using MTT assay it could be inferred that H. atra extracts can block the growth of
breast cancer cells (MCF-7) by inducing apoptosis. In Holothuria atra extract there is a possibility
of blocking the receptors such as the tyrosine kinases (RTKs) in cancer cells of Hep2, Hela and
143
MCF-7. The methanolic extracts of Holothuria atra showed maximum inhibition of antitumour
cells and it was observed by cell inhibition using tryphan blue.
Sulphorodamine B (SRB) assay
The cytotoxicity activity of S. pumila and H. atra extracts was carried out by SRB assay at
540nm. Inhibitory effects of S.pumila extracts revealed the reduction of growth in tumour cells
such as Vero, Hep2, Hela, MCF-7,cell lines. Cyclic α, β monoterpenoids compound and
flavonoids seen in Sigmadocia pumila could reduce the progression of tumour cells by arresting
the cell G2/M phase leading to apoptosis. Similarly aaptamine a compound of isoaaptamine
qunioline obtained from the sponge showed cytotoxicic effect against the murine and the human
cell lines (Shen et al., 1999). Bistheonellide swinholide a bioactive compound from sponge
Xestospongia sp (a Macrolide) with antitumour activity was detected by SRB assay (Saito et al.,
1998).
Similarly Petrocortynes derived from the sponge S. Pumila could be considered as a
potent growth inhibitors of all the human tumour cell lines. It is found that the antiproliferative
effects of S. pumila extracts lead to the process of apoptosis and necrosis in tumour cell lines.
Carboxymethyl nicotinic acid from sponge Anthosigmella raromicrosclera is a pyridine a
protease inhibitor (Matsunaga et al., 1998). Aragusterol A, a sponge steroid has been indicated as
an effective inhibitor of cell proliferation against the human and murine cancer cell panel in vitro
and in vivo assays targets the G1/S cell cycle phase (Fukuoka et al., 2000).
The antitumour properties seen in S. pumila would lead to the synthesis and biological
evaluation of various related analogs such as Prolylephedrine. It could have the ability to regulate
the enzymes causing tumour and inhibited the growth of Hela and MCF-7 cell lines.
Makaluvamines belonging to the chemical class of pyrroloiminoquinones, were first isolated from
marine sponges of the genera Zyzzya and Histodermella. These compounds were cytotoxic against
144
a number of cancer cell lines, such as MCF-7 which was explained by their DNA topoisomerase
II inhibiting activity (Carney et al., 1993). Considerable effect was seen in the Sigmadocia pumila
extract which inhibited the cell proliferation of Hep2 the maximum of 90% with minimum effect
seen in Vero cell lines at (75%). It could also be noted that the extracts of H. atra cause apoptosis.
It is initiated either at plasma membrane (extrinsic pathway) or within the cell (intrinsic pathway).
The alkaloid manzamine A, isolated from a variety of marine sponges, inhibited cell
migration of pancreatic cancer cells AsPC-1 in vitro and it also showed the potential
antimetastatic and proapoptotic effect on cancer cells (Guzman et al., 2010). From SRB assay it is
noted that the S. pumila extracts reduce the growth of tumour cell lines with 85% in Hela cell line
and 82% of MCF-7 cells. S. pumila have the possibilities to control and could reduce the
mechanism of metastasis in tumour cells.The elimination of tumour in the early stages is an
integral part of chemoprevention and measuring the cytotoxic properties of a given compound
from S. pumila and H. atra against cancer cells provides useful insight into its chemo protective
potential.
The branched chain fatty acid 12 methyl teradeconic acid extracted from the sea
cucumber (Cucumaria echinata) modulated PC3 cell growth of cancer cell lines. It also induced
apoptosis inhibited the progression of cancer cells (Yang et al., 2003). Hot water extract of sea
cucumber showed antitumour activity against human colon adenocarcinoma cells. It showed that
apoptosis was induced by the extracts in a dose dependent manner (Ogushi et al., 2006).
Adamantane and Propylparaben seen in the H. atra extracts could inhibit the growth of tumour
cells. Hence from the results of Sulphorodamine B assay suggested that the sea cucumber
Holothuria atra exhibited antitumour activity by triggering apoptosis. The apoptosis and growth
inhibitory activity could be considered as an important component for cancer chemopreventive
effects.
The Holothuria scabra extract showed effective viability against the Hela cell lines.
Xylosides of cholestanol and 7-sterols, predominant constituents in the steryl glycoside fraction
145
from the sea cucumber Eupentacta fraudatrix showed cytotoxic activities against the Hela cell
lines (Makarieva et al., 1993). Kumar et al., (1998) stated that new hydroxysterol glycosides
reported by the Indian scientists from the holothurian Synapta muculata displayed antitumour
activity. The antitumour activity of Holothuria atra was detected at the concentration of 0.78 mg-
10 mg/ml against the Hela cell lines. It showed inhibitory effect against the Hela cells at the range
of 88% which was considered as the maximum value detected in SRB assay.
Rodriguez et al . (1991) isolated five triterpene glycosides from the sea cucumber and
observed antitumor (antiproliferative) activities. As triterpene glycosides are water soluble, they
appeared to be high molecular weight polysaccharides or heat stable glycoproteins with
antitumour activity. The levels of total phenols and flavonoids from the Atlantic sea cucumber
were high in Cucumaria frondosa, showing strong growth inhibitory activity against the human
cancer cell lines (Mamelona et al., 2007). The H. atra collected from the south eastern region of
India have high rate of antitumour activity in Hep2 cell lines. However variations of antitumour
activity was detected in Holothuria atra among different cell lines in SRB assay and it was
compared with that of sponge S. pumila. Similar variations from the three sea cucumber species
Holothuria scabra, Holothuria leucospilota and Stichopus chloronotus against the cell lines A549
and C33A were demonstrated by using SRB assay. The variation in the activity among the species
was determined using SRB assay (Osama et al., 2009).
Observations made in this study showed that the Holothuria atra have the ability to
induce apoptosis and regulate the control in the tumor progressed cells. PE, a new sulfated
saponin compound derived from the sea cucumber Pentacta quadrangularis, was tested in a
number of in vitro assays to detect antitumor activity against cell division and vascularization.
All the standard methods for activity against endothelial cells including proliferation, apoptosis,
migration, attachment and tube formation were found to be positive with PE (Timothy et al.,
2005). The occurrence of various compounds in H. atra which include phenolic compounds,
isoflavonoids and glycosides indicated the process of antitumour activity. From the in vitro assays
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MTT and SRB, it is observed that the Holothuria atra extract could have acted as an initiator and
regulated the immune response against the cancer cells.
In vivo studies using DAL cells
Invivo studies of sponge S. pumila and H. atra in DAL induced Swiss albino mice
showed the antitumour property. The intraperitoneal inoculation of DAL cells in mice produced
increased proliferation of cells. Hematological parameters enabled to conclude the protective
effect of the extracts. Onnamide-A from marine sponge, Theonella spp. collected in Okinawa
showed in vitro cytotoxity and in vivo antitumour activity in many leukemia and solid tumour
model systems with increase in RBC count (Sakemi et al., 1988).
The compounds such as flavonoids and alkaloids in S. pumila could indicate the
reduction of growth in tumour cells. For similar observation in this study a DAL control group
was used. It showed reduced RBC count with increase in WBC count than normal group. The S.
pumila extract treated mice showed increase in RBC count and a decrease in elevated WBC count
and they were reported as confirmatory markers for the protection against DAL cells. Present
observations and comparisons suggest that Sigmadocia pumila and Holothuria atra possess potent
antitumour activity against the DAL cells.
Petrosaspongia mycofijiensis extract of 10mg/kg yielded mycothiazole a solid tumor
selective compound inhibited hypoxia-inducible factor-1 (HIF-1) signaling in tumour cells
(Morgan et al., 2010). Similarly S. pumila extracts were used at the concentration of 5 and
10mg/kg. The tumour volume seen in the mice was reduced at the level of 3.2±0.93 to 2.2±0.53
and showed significant activity (P < 0.01). An increase in the level of haemoglobin was observed
from 14.8±1.3 to 15.6±0.12. In S. pumila the packed cell volume (PCV) content decreased from
2.0±0.14 to 0.9±0.21. PCV in tumour control group increased significantly when compared with
normal group. Similarly the antitumour activity was detected using H. atra extracts in mice
against the DAL cell lines.
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Leucospilotaside A is the bioactive compound with potential cytotoxic effects identified
from H. leucospilota by in vivo analysis (Han et al., 2007). Presence of compounds such as 1, 2-
Benzisoxazole, isobenzofuran, and benzamidines could have acted as antitumour compounds in
reducing the growth of DAL cells. Thus by measuring the tumour volume, tumour cell count,
increased life span and hematological parameters significant antitumor activity could be
evaluated. The H. atra extract treated mice showed increase in RBC level and a decrease in WBC.
The tumour volume seen in H. atra was found to be from 2.40±0.11 to 1.01±0.04. The PCV was
noted as1.6±0.10 to 0.8±0.13. Intercedensides A, B, and C, and Intercedensides S2 were obtained
from sea cucumbers Holothuria nobilis and Merrsamaria intercedens. Such reductions in tumour
volume to the level of 2.2±0.42 to 1.8±0.86 showed significant activity (Wu et al., 2007).
Toxic triterpenoids obtained from sea cucumber Actinopyga agassizi were found to
inhibit the growth of sarcoma and adeno carcinoma in mice. Two saponins rhelothurium A and B
showed potent antitumor activity (Saki et al., 1996). The antiproliferative effect was seen in H.
atra against the DAL cell lines in mice with an increase in haemoglobin and RBC and a
concomitant decrease in WBC. Normally each mouse contains about 5 x 106
intraperitoneal cells,
of which 50% are macrophage (Natesan et al., 2007). H. atra extract treatment enhanced non
viable cell counts in peritoneal exudates. It is possible that absorption of H. atra extracts by viable
cells could lead to lysis of cell through activation of macrophages.
Filinopsides A and E from the sea cucumber Pentacta quadrangularis inhibited
angiogenesis in cultured human endothelial cells (HMECs) and inhibited the growth of mouse
Sarcoma- 180. It induced apoptosis in tumour and associated with tumour endothelial cells in vivo
and in vitro (Yi et al., 2006). The research results and the present data suggest that the Holothuria
atra could act as an inhibitor and have various growth regulating factors to control the abnormal
growth of tumor cells. The life span of mice increased to 66.0 and 75.0% in H. atra and S.pumila
extracts respectively. Thus S. pumila have more effect on DAL cells compared to that of H. atra
and could be further studied for their biomedical applications.
