Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines.
ABSTRACT Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.
- [show abstract] [hide abstract]
ABSTRACT: The DNA-damage response (DDR) pathways consist of interconnected components that respond to DNA damage to allow repair and promote cell survival. The DNA repair pathways and downstream cellular responses have diverged in cancer cells compared with normal cells because of genetic alterations that underlie drug resistance, disabled repair and resistance to apoptosis. Consequently, abrogating DDR pathways represents an important mechanism for enhancing the therapeutic index of DNA-damaging anticancer agents. In this review, we discuss the DDR pathways that determine antitumor effects of DNA-damaging agents with a specific focus on treatment outcomes in tumors carrying a defective p53 pathway. Finely tuned survival and death pathways govern the cellular responses downstream of the cytotoxic insults inherent in anticancer treatment. The significance and relative contributions of cellular responses including apoptosis, mitotic catastrophe and senescence are discussed in relation to the web of molecular interactions that affect such outcomes. We propose that promising combinations of DNA-damaging anticancer treatments with DDR-pathway inhibition would be further enhanced by activating downstream apoptotic pathways. The proposed rationale ensures that actual cell death is the preferred outcome of cancer treatment instead of other responses, including reversible cell cycle arrest, autophagy or senescence. Finally, to better measure the contribution of different cellular responses to anticancer treatments, multiplex in vivo assessments of therapy-induced response pathways such as cell death, senescence and mitotic catastrophe is desirable rather than the current reliance on the measurement of a single response pathway such as apoptosis.Oncogene 11/2010; 29(46):6085-98. · 7.36 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Tumor tissue is composed of both cancer cells and stromal cells recruited from normal tissue. These cells include fibroblastic cells, endothelial cells, and cells of hematopoietic origin. The host-derived stromal cells play a critical role in all aspects of cancer biology including transformation, progression, tumor growth, and drug resistance. The interactions between stromal cells and cancer cells are of intense interest, and their complex interactions are beginning to be identified. Therapies that target components of the tumor microenvironment are showing efficacy in the clinic, particularly when used in combination with other therapeutic agents. In general these agents have been well tolerated, and targeting the stromal components may be a strategy for circumventing the problem of drug resistance. In this review, we highlight major stromal components, their interactions with tumor cells, and therapeutic approaches that disrupt host-tumor cell interactions. Advances in understanding host stromal components with respect to origin, subsets, and their signaling networks will reveal novel targets. Synergistic approaches that disrupt multiple host-tumor cell signaling pathways will lead to more effective therapies for cancer.Current Opinion in Pharmacology 08/2010; 10(4):369-74. · 5.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Apoptosis as a form of programmed cell death is a critical defense mechanism against the formation and progression of cancer and exhibits distinct morphological and biochemical traits. In an in vivo situation, apoptosis functions to eliminate potentially deleterious cells without causing such adverse effects as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. Marine natural products have become an important source in the discovery of antitumor drugs, especially when modern technology makes it more and more feasible to collect organisms from seas. Although lack of an analog of a long ethno-medical history for finding clues, as compared with terrestrial habitats, still hinders the progress, an increasing number of compounds have been isolated from marine organisms that have been found to possess apoptosis-inducing and anticancer activities. This primer summarizes several such compounds, based on their effects on apoptotic signaling pathways, although most of these products have not yet been studied in depth for their mechanisms of action.Current drug targets 03/2010; 11(6):708-15. · 3.93 Impact Factor
Cacospongionolide and Scalaradial, Two Marine
Sesterterpenoids as Potent Apoptosis-Inducing Factors
in Human Carcinoma Cell Lines
Daniela De Stefano1*., Giuseppina Tommonaro2., Shoaib Ahmad Malik3, Carmine Iodice2, Salvatore De
Rosa2, Maria Chiara Maiuri1,3, Rosa Carnuccio1
1Dipartimento di Farmacologia Sperimentale, Facolta ` di Scienze Biotecnologiche, Universita ` degli Studi di Napoli Federico II, Napoli, Italy, 2National Research Council of
Italy-Institute of Biomolecular Chemistry, Pozzuoli (NA), Italy, 3INSERM U848, Institut Gustave Roussy, Villejuif, France
Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of
cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response
and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development
of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important
role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as
anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects
of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine
sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA
fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot.
The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116
(human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with
scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation.
Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased
expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells
overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early
apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-kB subunits, nuclear level. In
conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising
compounds to inhibit cancer cell proliferation.
Citation: De Stefano D, Tommonaro G, Malik SA, Iodice C, De Rosa S, et al. (2012) Cacospongionolide and Scalaradial, Two Marine Sesterterpenoids as Potent
Apoptosis-Inducing Factors in Human Carcinoma Cell Lines. PLoS ONE 7(4): e33031. doi:10.1371/journal.pone.0033031
Editor: Maurice A.M. van Steensel, Maastricht University Medical Center, The Netherlands
Received August 4, 2011; Accepted February 9, 2012; Published April 3, 2012
Copyright: ? 2012 De Stefano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no funding or support to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Cancer is a leading cause of death in industrialized countries
. Although mortality rates have declined in recent years due to
earlier detection and more options in treatment, most cancers
remain incurable. However, the increase of drug-resistant cancers
needs the identification of innovative drugs. It is worthy of note
that malignant cells are characterized by deregulated signalling
pathways involving proliferation, apoptosis, and angiogenesis
[2,3]. Apoptosis, a form of programmed cell death, is a critical
defense mechanism against the formation and progression of
cancer and exhibits distinct morphological and biochemical traits
. Apoptosis acts by eliminating potentially deleterious cells
without causing such adverse effects, as inflammatory response,
and ensuing scar formation. Therefore, targeting apoptotic
pathways becomes an intriguing strategy for the development of
chemotherapeutic agents .
In the last decades, marine natural products, and particularly
sesterterpenoids, have played an important role in the discovery
and development of new drugs for their wide variety of chemical
structures and biological activities . The richest marine source
of natural products has been soft-bodied organisms that lack
physical defences against their predators, and therefore rely on
chemical defence mechanisms involving cytotoxic secondary
metabolites . Interestingly, many of these compounds have a
strong potential as anticancer drugs and their most common
mechanisms reported are the inhibition of cell proliferation and/or
induction of cell death . It has been demonstrated that
scalaradial, extracted from Cacospongia scalaris, exerts an anti-
inflammatory effect by inhibiting PLA2 activity . Moreover, in
vitro studies reported that scalaradial inhibited EGF-stimulated Akt
 as well as NF-kB activation . Cacospongionolide, extracted
from Fasciospongia cavernosa, is known to inhibit NF-kB activation,
leading to an anti-inflammatory effect . Many evidences
PLoS ONE | www.plosone.org1 April 2012 | Volume 7 | Issue 4 | e33031
reported the anti-apoptotic role of NF-kB, which is persistently
activated in several malignancies .
In the present study, we examined the ability of scalaradial and
cacospongionolide to inhibit proliferation and induce apoptosis in
several human carcinoma cell lines.
Effect of Cacospongionolide and Scalaradial on Cell
The natural compounds scalaradial and cacospongionolide
were extracted from the sponges Cacospongia scalaris and Fascios-
pongia cavernosa, respectively, as previously reported [12,13]
(Figure 1). Incubation of T47D, A431, HCT116 and HeLa cells
with SC (1, 10, and 100 mg/ml) or CSP (1, 10, and 100 mg/ml) for
24 h caused a significant and concentration-dependent reduction
of cell viability (Figure 2A-D). The IC50 of SC were 0.024, 0.026,
1.851, 1.284 and 0.52 M for T47D, A431, HCT116, and HeLa ,
respectively whereas for CSP were 0.030, 0.089, 3.697, 4.302 and
4.49 M. Daunorubicin (100 mM) inhibited in a significant manner
cell viability (,20%, data not shown). DMSO did not modify cell
viability (,90%, data not shown).
Effect of SC and CSP on Cell Morphology
We investigated the effect of SC (10 mg/ml) or CSP (10 mg/ml)
on the morphologic changes visualised by phase-contrast micros-
copy of T47D human breast cancer cells after 24 h. The untreated
cells showed a normal morphologic aspect. Cells treated with SC
or CSP were suggestive of apoptosis by blebs recognition. Dauno
exhibited the same effect (Figure 2C).
Effect of SC and CSP on DNA Fragmentation
Internucleosomal DNA degradation determined qualitatively by
comet assay (Figure 3A) as well as by agarose gel electrophoresis
(Figure 3B) and quantitatively by the diphenylamine reaction
(Figure 3C) were analysed as parameters of apoptosis. The T47D
cells treated with SC (10 mg/ml), CSP (10 mg/ml) or Dauno
(100 mM) for 24 h were used to perform comet assay. Following
electrophoresis, the sample is stained with a DNA-binding dye and
viewed under a microscope. Short strands of DNA generated from
DNA strand breaks and/or alkaline labile sites migrate farther
than intact DNA during electrophoresis and form the ‘‘tail’’ of the
‘‘comet’’. As expected, SC, CSP and Dauno induced a significant
increase in DNA migration in human breast carcinoma cells, as
compared to control cells (Figure 3A). Moreover, the incubation of
T47D cells with SC (10 mg/ml) or CSP (10 mg/ml) for 24 h led to
the appearance of oligonucleosomal fragmentation with the
characteristic ladder pattern associated with apoptosis and elicited
a significant increase of DNA fragmentation (by 45.2761.8 and
39.4861.35%, respectively, n=3) as compared to control, the
untreated cells (3.1260.25%). Dauno similarly induced the same
effect (44.661.25%) (Figure 3B, C).
Effect of SC and CSP on Apoptotic Signalling Proteins
It has been described that a reduction in mitochondrial
transmembrane potential (DYm) precedes apoptosis and may
represent an early signalling event . Finally, we evaluated the
Figure 1. Cacospongionolide and Scalaradial: structures and original sponges. Chemical structures of cacospongionolide and scalaradial
isolated from marine sponges Fasciospongia cavernosa and Cacospongia scalaris, respectively.
Natural Products Inhibit Cancer Cell Proliferation
PLoS ONE | www.plosone.org2 April 2012 | Volume 7 | Issue 4 | e33031
effect of SC and CSP on the DYm. As shown in Figure 4A, two
different human carcinoma cell lines treated overnight with SC
and CSP showed a significant and concentration-dependent loss of
the mitochondrial transmembrane potential compared to untreat-
ed as well as vehicle-treated cells. CisPt exhibited the same effect
(Figure 4A). To investigate the involvement of apoptotic signalling
molecules following SC and CSP treatment, the expression level of
apoptosis related proteins were analyzed by means of an antibody
Figure 2. Effect of SC and CSP on cell viability and morphology. T47D (A), A431(B), HCT116(C) and HeLa (D) cells were incubated with SC (1,
10 and 100 mM) and CSP (1, 10 and 100 mM) for 24 h. Thereafter cell viability was determined by MTT assay as described in Materials and Methods.
Data are expressed as mean 6 S.E.M. of six separate experiments (***p,0.001 vs. untreated cells). T47D cells (E) were treated with SC (10 mg/ml), CSP
(10 mg/ml) or Dauno (100 mM) for 24 h, visualised by phase-contrast microscopy and photographed. The untreated cells (Control) exhibited normal
morphologic aspects, whereas the cells treated with CSP, SC and Dauno were suggestive of apoptosis. Data illustrated in (C) are from a single
experiment and are representative of three separate experiments.
Natural Products Inhibit Cancer Cell Proliferation
PLoS ONE | www.plosone.org3April 2012 | Volume 7 | Issue 4 | e33031
array. For this, HeLa cells were treated with SC (10 mg/ml) or
CSP (10 mg/ml) for 6 h. Cell lysates were prepared and apoptosis
antibody array was performed following the manufacturer’s
protocol. Our results showed that a number of apoptotic signalling
proteins were modulated following treatment of SC and CSP.
Among the pro-apoptotic proteins present on the array, a high
intensity of chemioluminescence was detected for cleaved caspase-
3. On the other hand, several anti-apoptotic proteins were
significantly inhibited by SC and CSP treatment, such as Survivin,
Bcl-2 and IAPs (Figure 4B). Surprisingly, SC and CSP elicited
similar changes on the expression level of proteins involved in the
apoptosis pathway (Figure 4B). However, often SC showed major
effects compared to CSP. Unfortunately, a co-treatment with SC
and CSP did not result in an addictive or synergistic effect (data
not shown). This kind of epistatic analysis confirms the pro-
apoptotic pathways elicited by both agents. In order to deepen our
results, we are further investigating the molecular mechanisms at
the basis of this expression pattern. Thus, HCT116 cells were
incubated with SC or CSP in the presence of the pan-caspase
inhibitor Z-VAD-fmk (50 mM) or the p53 inhibitor pifthrin-alpha
(30 mM). Results from FACS analysis demonstrated that SC as
well as CSP were not capable to cause cell death in the presence of
Z-VAD-fmk, thus suggesting that SC and CSP acted via caspase
(Figure 5). On the contrary, in the presence of pifthrin-alpha SC
and CSP caused a significant cell death, suggesting that p53 was
not involved (Figure 5). These results suggest that activated
caspases are key molecules to determine the effect of SC and CSP.
Effect of SC and CSP on p50 and p65 Nuclear
To explore whether the effects of SC and CSP on apoptotic
signalling proteins was attributable to NF-kB inhibition, we
evaluated T47D nuclear levels of p50 and p65, NF-kB subunits,
by Western blot. Treatment of cells with CSP (10 mg/ml) or SC
(10 mg/ml) for 24 h reduced nuclear translocation of both p50 (by
44.7265.73% and 29.4566.71%, respectively, n=3) and p65 (by
73.7761.51% and 80.4760.11%, respectively, n=3). Dauno
(100 mM) exhibited the same effects (by 30.2968.49% and
81.6360.46%, respectively, n=3) (Figure 6).
Cancer is a malignant disease resulting from several causes .
Among these, mutation of oncogenes, and/or tumor suppressor
genes can determine the alteration of signalling pathways,
including those involved in cancer cell proliferation . One of
the most important processes regulating the balance of cell growth
and cell death is apoptosis, toward which has been given much
attention for cancer treatment . Natural products, including
those of marine origin that show cancer-preventive and anticancer
properties, attract more attention as probable candidates to be
used in cancer preventive or chemotherapeutic strategies [18,19].
Cacospongionolide has been firstly reported to inhibit PLA2
activity in human neutrophils  and, subsequently, to reduce
NF-kB transcriptional activity in LPS-stimulated mouse peritoneal
macrophages . As for scalaradial, it has been reported to
inhibit PLA2 activity as well as EGF-stimulated Akt [7,8] and NF-
kB activation . NF-kB is persistently activated in several
malignancies and its anti-apoptotic role has been demonstrated
. The results of the present study indicate that scalaradial and
cacospongionolide are capable of causing cell death by inducing
apoptosis in four different human cancer cells. Preliminary, we
found that both scalaradial and cacospongionolide reduced human
cancer cell viability in a concentration-dependent manner, as
compared to untreated cells. In addition, scalaradial and
cacospongionolide induced a type of cell death that bears some
hallmarks of apoptosis, such as DYm dissipation and DNA
fragmentation, which were confirmed by optical microscopy,
Figure 3. Effect of SC and CSP on DNA fragmentation. The T47D
cells were treated with SC (10 mg/ml), CSP (10 mg/ml) or Dauno (100
mM) for 24 h, thereafter DNA fragmentation was analyzed by comet
assay (A), agarose gel electrophoresis (B) and diphenylamine assay (C).
Basal DNA damage from untreated cells, DNA damage from SC or CSP
cells (A). Data are from a single experiment and representative of 50
randomly selected cells stained with ethidium bromide (EtBr). Data
illustrated in (B) are from a single experiment and representative of
three separate experiments, while data in (C) are expressed as mean 6
S.E.M. of three separate experiments. ***p,0.001 vs. control (untreated
Natural Products Inhibit Cancer Cell Proliferation
PLoS ONE | www.plosone.org4April 2012 | Volume 7 | Issue 4 | e33031
comet assay and FACS. Progresses in cancer biology and genetics
have led to the concept that deregulation of apoptosis-related
proteins or genes bears prominent effect on the malignant
phenotype . In addition, it is widely demonstrated that some
oncogenic mutations suppressing apoptosis may lead to tumour
initiation, progression or metastasis . Therefore, apoptosis of
premalignant or malignant cells would represent a protective
mechanism against tumour formation and development. Interest-
ingly, conventional chemotherapy for cancer utilizes cytotoxic
agents that elicit their therapeutic effect partly through apoptosis
induction, whereas over-expression of anti-apoptotic proteins in
cancer cells can inhibit apoptosis and even engender chemoresis-
Figure 4. Effect of SC and CSP on apoptosis induction. Detection of dead and dying cells by FACS (A). Cells treated overnight with SC, CSP and
cisPt were stained with DYm-sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The white portions of the columns refer to the
DiOC6(3)low/PI+population (dead) and the remaining part of the column corresponds to the DiOC6(3)low/PI-(dying) population. Results are means 6
S.E.M. of three independent experiments. Expression profile of apoptosis-related proteins (B). Cell lysates from HeLa cells, treated with SC (10 mM) and
CSP (10 mM) overnight, were analyzed by human apoptosis profiler. Data are expressed as means 6 S.D. of two independent experiments.
Natural Products Inhibit Cancer Cell Proliferation
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tance . Furthermore, also radiation therapy is known to induce
apoptosis of malignant cells and an inherent resistance is mediated
by constitutively activated oncogenic, proliferative and anti-
apoptotic proteins/pathways . Therefore, chemotherapeutic
interventions often fail to determine complete health in patients.
Based on this knowledge, it would be useful to have new drugs
overcoming the impact of resistance and increase the anticancer
efficacy. Recently, many anti-tumour agents target proteins
involved in apoptosis while the effects of these agents might be
various, and they either induce cancer cell death or enhance the
sensitivity of cancer cells to certain cytotoxic drugs or radiation
[24,25]. Here, we have shown that scalaradial and cacospongio-
nolide exert a significant effect on inhibition of cell growth and
induction of apoptosis in four different human tumoral cells by
induction of the apoptotic pathway. Particularly, anti-apoptotic
proteins were significantly inhibited by scalaradial and cacospon-
gionolide treatment, such as Survivin, Bcl-2 and IAPs. Interest-
ingly, SC or CSP in the presence of the pan-caspase inhibitor Z-
VAD-fmk were not capable to cause cell death. In contrast, the
p53 inhibitor did not modified the effect of SC and CSP, thus
suggesting that SC and CSP did not acted via p53. These results
suggest that activated caspases are key molecules to determine the
effect of SC and CSP. Thus, the significant reduction of several
anti-apoptotic proteins expression suggests promising anticancer
properties for both compounds. It is well known that many pro-
and anti-apoptotic proteins are regulated at transcriptional level by
NF-kB . Here, we reported that scalaradial and cacospongio-
nolide were capable of inhibiting p50 and p65 nuclear
translocation. However, further investigations are necessary for a
more detailed understanding of the molecular mechanisms by
which cacospongionolide and scalaradial induce cancer cell
apoptosis. In conclusion scalaradial and cacospongionolide, by
determining human cancer cell apoptosis, may represent a new
promising compounds to inhibit cancer cell proliferation.
Materials and Methods
No specific permits were required for the described field studies.
No specific permissions were required for collecting these sponges
because the location they were taken from is not privately-owned
or protected in any way. In addition, this study does not involve
endangered or protected species.
Figure 5. Effect of SC and CSP on cell viability. FACS analysis
shows the effect of SC (10 mg/ml) or CSP (10 mg/ml) on HCT116 cell
viability in the presence or absence of Z-VAD-fmk (50 mM) and pifthrin-
alpha (30 mM). Data are the mean 6 SD of three experiments in
Figure 6. Effect of SC and CSP on p50 and p65 nuclear translocation. Representative Western blot of p50 and p65 as well as the
densitometric analysis shows p50 and p65 nuclear translocation in T47D cells incubated with CSP (10 mg/ml) or SC (10 mg/ml) for 24 h. The results are
from a single experiment and are representative of three separate experiments. Densitometric data are expressed as mean 6 S.E.M. of three separate
experiments. ***p,0.001 vs. untreated cells. The expression of GAPDH is shown as an equal loading control.
Natural Products Inhibit Cancer Cell Proliferation
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Isolation of Scalaradial and Cacospongionolide
Scalaradial (SC) was isolated from the sponge Cacospongia scalaris,
while Cacospongionolide (CSP) was isolated from the sponge
Fasciospongia cavernosa. Both sponges were collected in Rovijni
(Croatia) in summer 2007, and kept in freezer at -20uC until
analysis. The isolation of SC and CSP was performed as previously
T47D (breast carcinoma), A431 (epidermoid carcinoma),
HeLa (cervix adenocarcinoma) human cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
fetal calf serum (FCS), 1 mM pyruvate and 10 mM Hepes at
37uC under 5% CO2. HCT116 human colon carcinoma cells
were cultured in McCoy’s 5A medium containing 10% FCS,
100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 U/ml
penicillin G sodium, and 100 mg/ml streptomycin sulfate (37uC,
5% CO2). All media and supplements for cell culture were
purchased from Gibco-Invitrogen (Carlsbad, USA). Cells (36105)
were seeded in 24-well plates and grown for 24 h before
treatment with scalaradial (SC at a final concentration/per well
1, 10 and 100 mg/ml, which is 0.0026, 0.026 and 0.26 M,
respectively), cacospongionolide (CSP at a final concentration/
per well 1, 10 and 100 mg/ml which is 0.0025, 0.025 and
0.25 M, respectively), daunorubicin (Dauno at a final concen-
tration/per well 100 mM) or cisPlatinum (cisPt at a final
concentration/per well 7.5, 15, 20 and 40 mM) for the indicated
period. Cell lines were all from ATCC catalogue. In some
experiments, cells were incubated with the pan-caspase inhibitor
Z-VAD-fmk (50 mM) or the p53 inhibitor pifthrin-alpha (30 mM)
in the presence of SC or CSP.
MTT Viability Assay
The cells were plated in 96 culture wells at a density of 250.000
cells/ml and allowed to adhere for 2 h. Thereafter the medium
was replaced with fresh medium and cells were incubated with SC,
CSP or Dauno alone or in association. After 24 h, cell viability was
determined by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) conversion assay . Briefly, 125 ml
of MTT (5 mg/ml in complete DMEM) were added to the cells
and incubated for additional 3 h. After this time point the cells
were lysed and the dark blue crystals solubilized with 375 ml of a
solution containing 50% (v:v) N,N-dymethylformamide, 20% (w:v)
SDS with an adjusted pH of 4.5. The optical density (OD) of each
well was measured with a microplate spectrophotometer (Titertek
Multiskan MCCC/340) equipped with a 620 nm filter. The cell
viability in response to treatment with test compounds was
calculated as % cell viability=(OD treated/OD control)6100.
T47D cells (16106cells/ml) were plated in 35610 mm
diameter culture dishes and incubated with SC (10 mg/ml), CSP
(10 mg/ml) or Dauno for 24 h. Thereafter, the cells were washed
three times with PBS, fixed with 3% formaldehyde in PBS, and
then visualised by phase-contrast microscopy (12,5x; LAS, Leica).
The comet assay was performed according to a standard
protocol . Aliquots of 5 ml cell suspension (about 15000
T47D cells treated as described above) were mixed with 120 ml
low melting point agarose (0.5% in phosphate-buffered saline)
and added to microscope slides, which had been covered with a
bottom layer of 1.5% agarose. Slides were lysed (pH 10; 4uC)
denaturation of 25 min and electrophoresis (0.86 V/cm) of 25
min at a pH.13. Slides were washed three times in Tris/
EDTA (TE) buffer (100 mM Tris, 5 mM EDTA, pH 10). After
removing the coverslips, slides were processed as usual. Slides
were coded and images of 50 randomly selected cells stained
with ethidium bromide (EtBr) were analysed from each slide
and visualized by fluorescence microscopy (5x; LAS, Leica).
at least 1 h and processedusinga time of alkali
Gel electrophoresis of Fragmented DNA
T47D (26106) cells were seeded on 10 cm diameter Petri dishes
and allowed to adhere for 2 h. Thereafter the medium was
replaced with fresh medium and the cells were incubated as
described above for 24 h. For analysis of genomic DNA, cells were
gently scraped off and collected together with non-attached cells in
the supernatant. Cells were resuspended in 250 ml of Tris-Borate
EDTA (TBE) buffer containing 0.25% NP-40 and 100 mg/ml
RNAse A. After incubation at 37uC for 30 min., extracts were
treated with 1mg/ml proteinase K for additional 30 min. at 37uC.
Then equal amounts (10 ml) of the extracts were loaded on a 1%
agarose gel containing (1 mg/ml) ethidium bromide and run for 1h
in 16TBE at 90 V. The gel captions were obtained with Polaroid
Quantification of DNA Fragmentation
DNA fragmentation was performed essentially as previously
described by Messmer and Brune  with some modification.
Briefly, T47D cells (46106) were seeded on 10 cm diameter
culture dishes and incubated with CSP, SC or Dauno for 24 h.
Thereafter, the cells were scraped off the culture plates,
centrifuged at 1806g for 10 min, resuspended in 250 ml of TE-
buffer (10 mM Tris, 1 mM EDTA, pH 8.00), and incubated
with an additional volume of lysis buffer (5 mM Tris, 1 mM
EDTA, pH 8.00, 0.5% Triton X-100) for 30 min at 4uC. After
lysis, the intact chromatin (pellet) was separated from DNA
fragments (supernatant) by centrifugation for 15 min at 13
000 rpm. Pellet was resuspended in 500 ml of TE-buffer and
samples were precipitated by adding 500 ml of 25% trichlor-
oacetic acid at 4uC. Samples were pelleted at 30006g for 10 min
and the supernatant was removed. After addition of 300 ml of
5% trichloroacetic acid, samples were boiled for 15 min. DNA
contents were quantified by using the diphenylamine (DPA)
reagent (0.3 g DPA, 0.2 ml of sulphuric acid, 0.1 ml of 1.6%
acetaldehyde, 10 ml of acetic acid glacial). The percentage of
fragmented DNA was calculated as the ratio of the DNA content
in the supernatant to the amount in the pellet.
The following fluorochromes were employed to determine
apoptosis-associated changes by cytofluorometry: 3,39-dihexylox-
acarbocyanine iodide (DiOC6(3), 40 nM) for quantification of
the mitochondrial transmembrane potential (DYm) and propi-
dium iodide (PI; 1 mg/ml) (both from Molecular probes) for
determination of cell viability . HeLa and HCT116 cells
were trypsinized and labeled with the fluorochromes at 37uC,
followed by cytofluorometric analysis with a fluorescence-
activated cell sorter (FACS) scan (Becton Dickinson).
The expression profile of apoptosis-related proteins was
detected and analyzed using a human apoptosis array kit
(ARY009), according with the manufacturer’s instructions (R&D
Systems). Briefly, the membrane containing immobilized apopto-
Natural Products Inhibit Cancer Cell Proliferation
PLoS ONE | www.plosone.org7April 2012 | Volume 7 | Issue 4 | e33031
sis-related antibodies was blocked with bovine serum albumin for
1 h on a rocking platform at room temperature. The membrane
was then incubated with lysates of untreated or treated HeLa cells
(for 6h with SC, 10 mg/ml, or CSP, 10 mg/ml) along with
Detection Antibody Cocktail overnight at 2uC to 8uC on a rocking
platform. The membrane was incubated with streptavidin-
horseradish peroxidase conjugate followed by chemiluminescent
detection reagent. The membrane was scanned and pixel density
was presented by quantifying the mean spot densities from two
separate experiments. For quantification, the spot volume was
determined, corrected for background and expressed as fold
change (treated vs. untreated cells).
Western Blot Analysis
Cytosolic and nuclear fraction proteins from T47D cells
incubated for 24 h as described above were prepared as previously
described  and mixed with gel loading buffer (50 mM Tris,
10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/ml of
bromophenol) in a ratio of 1:1, boiled for 3 min and centrifuged at
10,000 g for 5 min. Protein concentration was determined and
equivalent amounts (30 mg) of each sample were electrophoresed
in a 8–12% discontinuous polyacrylamide minigel. The proteins
were transferred onto nitro-cellulose membranes, according to the
manufacturer’s instructions (Protran, Schleicher & Schuell, Dassel,
Germany). The membranes were saturated by incubation at room
temperature for 2h with 10% non-fat dry milk in phosphate buffer
saline (PBS) and then incubated with (1:1000) anti-p50 or anti-p65
at 4uC overnight. The membranes were washed three times with
0.1% Tween 20 in PBS and then incubated with anti-rabbit, anti-
mouse or anti-goat immunoglobulins coupled to peroxidase
(1:1000) (DAKO, Milan, Italy). The immunocomplexes were
visualised by the ECL chemiluminescence method (Amersham,
Milan, Italy). The membranes were stripped and re-probed with
GAPDH antibody to verify equal loading of proteins. Subse-
quently, the relative expression of p50 and p65 proteins in nuclear
fractions was quantified by densitometric scanning of the X-ray
films with a GS 700 Imaging Densitometer and a computer
programme (Molecular Analyst, IBM).
Results are expressed as the means 6 SEM of n experiments.
Statistical significance was calculated by one-way analysis of
variance (ANOVA) and Bonferroni-corrected P-value for multiple
comparison test. The level of statistically significant difference was
defined as p,0.05.
Conceived and designed the experiments: DDS GT SAM MCM SDR RC.
Performed the experiments: DDS GT CI SAM MCM. Analyzed the data:
DDS GT SAM CI MCM SDR RC. Contributed reagents/materials/
analysis tools: DDS GT SAM MCM SDR RC. Wrote the paper: DDS GT
SAM MCM SDR RC. Obtained permission for use of cell line: RC.
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