Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines.

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.


Introduction
Cancer is a leading cause of death in industrialized countries [1]. 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 [4]. 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 [4].
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 [5]. 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 [6]. 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 [4]. It has been demonstrated that scalaradial, extracted from Cacospongia scalaris, exerts an antiinflammatory effect by inhibiting PLA2 activity [7]. Moreover, in vitro studies reported that scalaradial inhibited EGF-stimulated Akt [8] as well as NF-kB activation [9]. Cacospongionolide, extracted from Fasciospongia cavernosa, is known to inhibit NF-kB activation, leading to an anti-inflammatory effect [10]. Many evidences reported the anti-apoptotic role of NF-kB, which is persistently activated in several malignancies [11].
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 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 microscopy 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 (DY m ) precedes apoptosis and may represent an early signalling event [14]. Finally, we evaluated the effect of SC and CSP on the DY m . 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 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 proapoptotic 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 Translocation
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).

Discussion
Cancer is a malignant disease resulting from several causes [15]. Among these, mutation of oncogenes, and/or tumor suppressor genes can determine the alteration of signalling pathways, including those involved in cancer cell proliferation [16]. 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 [17]. 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 [12] and, subsequently, to reduce NF-kB transcriptional activity in LPS-stimulated mouse peritoneal macrophages [10]. As for scalaradial, it has been reported to inhibit PLA2 activity as well as EGF-stimulated Akt [7,8] and NF-kB activation [9]. NF-kB is persistently activated in several malignancies and its anti-apoptotic role has been demonstrated [11]. 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 DY m dissipation and DNA fragmentation, which were confirmed by optical microscopy, 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 [20]. In addition, it is widely demonstrated that some oncogenic mutations suppressing apoptosis may lead to tumour initiation, progression or metastasis [21]. Therefore, apoptosis of premalignant or malignant cells would represent a protective mechanism against tumour formation and development. Interestingly, 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- tance [22]. Furthermore, also radiation therapy is known to induce apoptosis of malignant cells and an inherent resistance is mediated by constitutively activated oncogenic, proliferative and antiapoptotic proteins/pathways [23]. 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 cacospongionolide 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 cacospongionolide treatment, such as Survivin, Bcl-2 and IAPs. Interestingly, 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 proand anti-apoptotic proteins are regulated at transcriptional level by NF-kB [26]. Here, we reported that scalaradial and cacospongionolide 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.

Ethics Statement
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.  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 described [12,[13][14][15][16][17][18][19][20][21][22][23][24][25][26].

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-2Htetrazolium bromide (MTT) conversion assay [27]. 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.

Comet Assay
The comet assay was performed according to a standard protocol [28]. 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) for at least 1 h and processed using a time of alkali 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).
Gel electrophoresis of Fragmented DNA T47D (2610 6 ) 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 T667.

Quantification of DNA Fragmentation
DNA fragmentation was performed essentially as previously described by Messmer and Brune [29] with some modification. Briefly, T47D cells (4610 6 ) 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 TEbuffer (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% trichloroacetic 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.

Apoptosis Array
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-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 streptavidinhorseradish 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 [27] 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, antimouse 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. Subsequently, 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).

Statistical Analysis
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.