Sarcophine-Diol Inhibits Expression of COX-2, Inhibits Activity of cPLA2, Enhances Degradation of PLA2 and PLCγ1 and Inhibits Cell Membrane Permeability in Mouse Melanoma B16F10 Cells

Sarcophine-diol (SD) is a semi-synthetic derivative of sarcophine with a significant chemopreventive effect against non-melanoma skin cancer both in vitro and in vivo. Recently, we have studied the effect of SD on melanoma development using the mouse melanoma B16F10 cell line. In this study, our findings show that SD suppresses cell multiplication and diminishes membrane permeability for ethidium bromide (EB), a model marker used to measure cell permeability for Ca2+ ions. SD also decreases protein levels of COX-2, and increases degradation of phospholipases PLA2 and PLCγ1 and diminishes enzymatic activity of the Ca2+-dependent cPLA2. This lower membrane permeability for Ca2+-ions, associated with SD, is most likely due to the diminished content of lysophosphosphatidylcholine (lysoPC) within cell membranes caused by the effect of SD on PLA2. The decrease in diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) due to inhibition of PLCγ1, leads to the downregulation of Ca2+-dependent processes within the cell and also inhibits the formation of tumors. These findings support our previous data suggesting that SD may have significant potential in the treatment of melanoma.


Introduction
Recently, there has been a considerable interest in the use of natural marine products for inhibiting skin cancer development [1][2][3][4][5][6][7]. In the last decade, our laboratory has taken the lead to study chemopreventive skin cancer as well as the anti-cancer effects of sarcophine diol (SD). SD ( Figure 1) is a semi-synthetic derivative of sarcophine which is a cembranolide isolated from the Red Sea coral Sarcophyton glaucum [3]. Our studies demonstrated that SD is a very promising chemopreventive agent against chemically-induced and UV-induced non-melanoma skin cancer in both animal and cell-culture models. Topical application of SD was found to enhance expression levels of cleaved-Caspase-3 and -8, molecular markers for apoptosis in chemically-induced skin cancer in female CD-1 mice [1]. We also demonstrated that SD treatment inhibits skin tumor development in UV-induced skin cancer in SKH-1 hairless mice [4]. The latter investigations also revealed that topical application of SD enhances cellular levels of cleaved-Caspase-3 and -8, and increases the rates of DNA fragmentation in skin cells isolated from these mice [4]. Our subsequent studies demonstrated that SD, in a concentration-dependent manner, decreases cell viability in human epidermoid carcinoma A431 cell line [8]. These studies also showed that SD treatment inhibits the incorporation of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) in de novo synthetized DNA. Moreover, SD treatment was found to enhance fragmentation of DNA in the same human epidermoid carcinoma A431 cell line and increases expression level of caspase-3 through activation of upstream caspase-8 in these cancer cells [8].
Recently, we extended our research to study the effect of SD on melanoma development using the mouse melanoma B 16 F 10 cell line since numbers of new melanoma diagnoses has been on the rise [9]. Our results showed that SD inhibits the de novo DNA synthesis and enhances DNA fragmentation. SD also inhibits the expression of several biomarkers of proliferation and apoptosis [10]. It inhibits the levels of signal transducer and activator of transcription protein STAT-3 and cyclin D1, an activator of cyclin-dependent kinase 4 (cdk4), enhances the levels of tumor suppressor protein p53, and stimulates cleavage of the nuclear poly-(ADP-ribose) polymerase PARP. It also enhances both protein levels as well as the enzymatic activities of caspase-3, -8 and -9. These findings, in addition to inhibition of cell viability, suggest that SD inhibits melanoma cell proliferation by arresting the cell-division cycle in the

SD Inhibits Cell Multiplication
As illustrated in Table 1, the total living cell numbers in untreated control cell samples increased by ~13.7-fold during 72 h incubation period. The total protein content in untreated control cells increased by ~11.6-fold during 72 h incubation. Treatment of the same cells with increasing concentrations of SD inhibits cell duplication in a dose-dependent manner. This is reflected in the total number of living cells and the total cellular protein content. Maximal inhibition of cell growth in terms of both total living cell numbers and total cellular protein content compared to untreated controls was observed in cells treated with 250 µM concentration of SD for 72 h of treatment. To illustrate the inhibitory effect of SD treatment on melanoma cell multiplication, cell extracts were loaded on nitrocellulose membranes and stained with Amido-black. Figure 3 shows images from the attached cell samples loaded from the same concentration. Protein content in cells treated with a 250 µM concentration of SD for 72 h is nearly the same as untreated control cells at time "0", suggesting that SD most likely inhibits wound healing due to inhibition of cell multiplication, rather than directly inhibiting the invasiveness of the cells.

SD Inhibits Cell Membrane Permeability
The effect of SD on membrane permeability was studied. It is commonly accepted that free levels of Ca 2+ ions within the cytoplasm play a crucial role in the regulation of several vital processes [13,14].  As cPLA 2 catalyzes hydrolysis and release of arachidonic acids from position C-2 in esterified PC [26], leading to elevation of lysophosphatidylcholine (lysoPC) in the cell membrane [27], the effects of SD treatment on enzymatic activity of this phospholipase was investigated. Using the synthetic substrate arachidonyl thio-PC and specific inhibitors for iPLA 2 and sPLA 2 , the enzymatic activity of cPLA 2 in protein extracts from untreated control cells and cells treated with SD for 24 h and 72 h was determined. As illustrated in Figure 6, exposure of the cells to a 250 µM concentration of SD produces a time-dependent decline in enzymatic activity of cPLA 2 . The lowest enzymatic activity (70%), measured as rate of hydrolysis of arachidonyl thio-PC by cPLA 2 , was observed at a 250 µM concentration of SD and 24 h of incubation. Further incubation time does not decrease the activity any further.

Discussion
In our recent study [10], SD was found to inhibit DNA synthesis, determined by incorporation of BrdU on de novo synthetized DNA, and simultaneously stimulate DNA fragmentation in B 16 F 10 melanoma cells. SD was also found to enhance degradation of PARP that appears to be involved in

Time of cells treatment [hours]
DNA repair and helps cells to maintain their viability [28]. Inactivation of PARP can be facilitated by many ICE-like caspases, and PARP is one of the main cleavage targets for Caspase-3 in situ [29][30][31][32][33][34]. The same study also demonstrated that SD treatment increases the protein level of cleaved-Caspase-3 and its enzymatic activity. As a result, cells treated with SD are characterized as having lower content of intact-PARP and higher content of the cleaved-PARP isoform, as compared to untreated control cells. Data also shows that SD inhibits expression of STAT-3, a protein that plays a role in the expression of genes that are involved in cell survival, proliferation, chemo-resistance and angiogenesis [35,36]. Moreover, SD treatment was also demonstrated to increase expression of the tumor suppressor protein p53, which is known to activate many pro-apoptotic factors, including BAX, Bak, Fas/APO, PiDD, etc. [37,38].
To extend our studies to further explore the inhibitory effect of SD on melanoma and to identify a possible mechanism of action at the molecular level, our investigation showed that SD inhibits cell duplication. This notion is based on findings that cells treated with SD display the same fraction of floating cells/dead cells as untreated control cells.
Treatment with SD inhibits staining of our cells with EB suggesting that it inhibits permeability to Ca 2+ and thus inhibits Ca 2+ -dependent processes within cells. It is commonly accepted that cyclooxygenase-2 (Cox-2) is undetectable in most normal tissues. It is an inducible enzyme, becoming abundant at sites of inflammation [20]. Recently, it has been shown to be upregulated in various carcinomas [21,22]. In this investigation, SD was found to decrease cellular levels of Cox-2. Cox-2 converts arachidonic acid that is released by cPLA 2 from phospholipids, particularly phosphatidylcholine (PC) [16,17] to prostaglandin H2 (PGH2), a precursor of the series-2 prostanoids [18,19]. SD treatment stimulates inactivation of cPLA 2 . High activity of cPLA 2 in tumor cells [11] increases levels of lysoPC within the cell membrane. Several studies have demonstrated that a higher content of lysoPC makes plasmalemma more fluid [27] and permeable to intracellular mediators such as Ca 2+ ions [39,40]. Membrane permeability is implicated in DNA damage and repair [41,42] and there is growing evidence showing that high content of lysoPC accompanies inflammation [21,22], cell proliferation [43,44] and cancer development [11,45].
Untreated control tumor cells display high activities of cPLA 2 and PLC γ 1. High activity of cPLA 2 increases cell membrane fluidity due to higher content of lysoPC in membranes and causing free Ca 2+ ions concentration in the cytoplasm to rise. Higher content of arachidonic acid induces activity of Cox-2 and the production of prostaglandins. Higher activity of PLC γ 1 produces DAG, an activator of protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP 3 ). The IP 3 activates Ca 2+ ion channels in the endoplasmic reticulum that enhances the level of free Ca 2+ ions in the cytoplasm [46][47][48]. SD was found to stimulate degradation and cleavage of PLC γ 1 and cPLA2. This suggests that SD is involved in the regulation of two important signaling mediators derived from membrane-associated phosphatidylinositol 4,5-biphosphate (PIP 2 ): diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ) that play a role in several biochemical processes. DAG acts similarly to phorbol esters, which are well known tumor promoters that activate protein kinase C (PKC) cascades [31,32] and was found to be a strong enhancer of cell proliferation [46]. PKC signaling pathways are involved in phosphorylation of many proteins in vitro and are upregulated in different types of cancers including cutaneous melanoma [46]. Thus the fact that SD stimulates cleavage and degradation of PLC γ 1 suggests that SD also inhibit the synthesis of an endogenous tumor-promoting factor (DAG).

Chemical and Reagents
The gel apparatus and material for electrophoresis were purchased from Hoefer (San Francisco, CA) and Bio-Rad (Richmond, CA). All commonly used reagents, 10,000 units of Penicillin, 10 mg Streptomycin stock solutions and Trypsin-EDTA 10× were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor488-phalloidin was purchased from Invitrogen (Eugene, CA). 0.4% Trypan-blue solution was purchased from MidSci Amersco (Solon, OH) and 4% paraformaldehyde in PBS buffer solution was purchased from USB Corporation (Cleveland, OH). The Elisa-type kit for determination of enzymatic activity of cPLA 2 was purchased from Cayman Chemical Company (Ann Arbor, MI). Mouse melanoma B 16

Semi-Synthesis of Sarcophine-Diol (SD)
Sarcophine was isolated from the Red Sea soft coral Sarcophyton glaucum as described previously [3,4], and converted to sarcophine-diol (SD) using a two-step reaction according to our published procedures [3]. SD was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. In all culture media, the final concentration of DMSO was 0.5% including untreated controls, whereas the final concentration of SD ranges from 0 µM to 250 µM.

Cell Culture
Under standard conditions, cells were grown in DMEM media supplemented with 10% FBS and 1% solution containing 100 U of penicillin/mL of media and 100 mg streptomycin/mL of media. Cells were incubated in 37 °C in a humidified atmosphere containing 95% O 2 and 5% CO 2 .

Wound Healing Assay
A wound healing assay was used to compare the relative proliferation of control cells and cells treated with SD. ~7 × 10 4 cells were seeded in growth media in a polystyrene petri dish (3.5 cm diameter) and incubated for 24-36 h until cells completely covered the surface of the dish. A wound of <1 mm in width was made across the dish using a rubber scraper, and the scraped/detached cells were gently removed from the dish. Fresh media was poured into each dish, and cells were incubated in the presence of a 250 µM concentration of SD for 24-96 h. A control experiment without SD was also carried out. After each 24 h cycle, wound healing was examined using light microscopy.

Cell Membrane Permeability
Ethidium bromide (EB) was used to assess the membrane permeability for Ca 2+ -ions. ~2-4 × 10 3 cells suspended in 0.75 mL of DMEM growth media were seeded in 24 well plates with removable glass cover-slips. Cells were incubated for 24 h. Once cells became attached to the surface, the old media was exchanged with fresh media containing 0.5% DMSO (control cells) or SD (250 µM), and cells were incubated for 24 h and 72 h. At the end of incubation period, cells were quickly washed with PBS (3 × 0.5 mL each time), and then stained with EB in 0.5 mL PBS (10 µg EB per mL, final concentration) at room temperature for 15 min with constant gentle rocking. Cells were then thoroughly washed with PBS to remove the excess of dye (4 times with 1 mL PBS, 5 min each). Cells were removed from the well and the cover-slips were mounted on a specimen glass slide. The EB stained nucleic acids were examined under a model A × 10 Zeiss fluorescence microscope (Jena, Germany) connected to AxioVison Rel.4.7 software via a Zeiss camera model AxioCam MRC5.

Determination of Cell Multiplication and Cellular Protein Content
A fixed number of cells were seeded in each well of a 6 well plate (~0.8 × 10 5 cells/5 mL well), and incubated for 24 h. Upon removal of the old media, cells in fresh media were incubated in the presence and absence of different concentrations of SD ranging from 0 µM to 250 µM for 72 h. Then, both the growth media and the attached cells were collected, and these two cell-containing fractions were used for determining the total cell number and also for total cellular protein contents.
To determine total protein contents and to perform Western blot analysis of Cox-2 and PARPs, cells were detached from the surface of a polysterene flask by short exposure to a minimal concentration of trypsin. Immediately after detachment, the cell suspension was diluted with 10-fold excess of growth media to inhibit further proteolysis by trypsin. To determine the status of degradation of PLA 2 and PLC γ 1 as well as enzymatic activity of cPLA 2 in cellular extracts, cells were detached from the flask using a rubber scraper. The latter procedure minimizes cleavage of these phospholipases by cellular proteases, including Caspases [11,12]. Immediately after detachment, the cells were pelleted in 4 °C at 1700 rpm for 5 min. Then, the cell pellets were thoroughly washed with PBS to remove contamination from FBS, and this procedure was repeated 3 times. The final cell pellets were solubilized in 3-5 volume of the appropriate ice-cold extraction buffer by passing through a pipette tip several times. Cell lysates were then kept in an ice-cold extraction buffer for 10-15 min, and spun down at 4 °C at 14,000 rpm for 20 min. Supernatants were collected and stored at −70 °C for further determinations.

Western Blot Analysis
A total cell protein extracts (15-20 µg protein/well) were resolved in 8.5% SDS-PAGE (having a total monomer to cross-linker ratio of 29:1). Once separated, proteins were transferred in an electric field into a nitrocellulose membrane and identified by the protein anti-bodies using the following procedure: The nonspecific binding sites were blocked by Tris-buffered saline pH 7.5 supplemented with 0.05% polyoxyethylenesorbitol monolaurate (Tween 20) and 5% non-fat milk at room temperature for 1 h. Then, membranes were probed with primary antibodies at 4 °C overnight using a dilution of 1:1000 for β-actin, Cox-2 and PARPs, and 1:500 for PLA 2 and PLC γ 1. Primary antibodies were diluted in Tris-buffer saline pH 7.5 containing 0.05% Tween and 1% non-fat milk. Membranes were thoroughly washed with Tris-buffer saline pH 7.5 containing 0.05% Tween 20, and then secondary antibodies were applied. All secondary antibodies were diluted in Tris-buffer saline supplemented with 0.05% Tween 20, using a dilutions of 1:2000 for detection of β-actin, Cox-2 and PARPs, and 1:1000 for PLA 2 and PLC γ 1. Membranes were exposed to secondary antibodies at room temperature for 1.5 h. After several washes with Tris-buffer saline pH 7.5 the protein bands were visualized by the ELC detection kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and quantified using UVP Biochem Gel Documentation System (UVP, Inc., Upland, CA). Equal protein loading was ensured by re-probing each membrane with anti-β-actin antibody.

Determination of Enzymatic Activity of cPLA 2
An Elisa-type kit (Cayman Chemical Company, Ann Arbor, MI) was used to determine enzymatic activity of the Ca 2+ -dependent phospholipase A 2 (cPLA 2 ) in the protein extracts from cells that grow in the presence and absence of SD. Determination of cPLA 2 activity was based on detection of free thio-arachidonic acid released from the synthetic arachidonyl thio-PC by cPLA 2 using Ellman's reagent. To inhibit the Ca 2+ -independent PLA 2 (iPLA 2 ) and the secretory PLA 2 (sPLA 2 ) activities, all reaction mixtures were supplemented in a 5 µM concentration of bromophenol lactone (BEL) and a 3 µM of thioetheramide-PC, respectively. Cellular protein extracts in concentrations of 3.5 mg/mL in 0.2 mL of reaction buffer containing arachidonyl thio-PC (50 µM final concentration) were incubated at room temperature for 1 h. The reaction was stopped by adding 5,5′-dithio-bis-(2-nitrobenzoic acid) (DNTB) in the EGTA/Tris-HCl buffer solution to the incubation mixture. Absorbance of the samples was read at 405 nm. The manufacturer assay buffer consisted of: 80 mM 4-(2-hydroxyethyl)piperazine-1-ethansulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 10 mM CaCl 2 , 4 mM Triton X-100, 30% glycerol, and 1 mg/mL BSA.

Staining of the Cells with Trypan-Blue
A quick Trypan-blue exclusion staining method was also used to determine cell viability. Cell suspension in growth media was diluted 2-fold with a 0.4% Trypan-blue solution. The number of living cells were determined using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience LLC, Lawrence, MA) equipped with Cellometer Automated Cell Counts software.

Determination of Protein Content
Total protein content in the cell extracts was determined colorimetrically using a detergent compatible bicinchoninic acid (BCA) protein assay kit according to the manufacturer protocol (Pierce, BCA, Rockford, IL) using bovine serum albumin (BSA) as a standard.

Statistical Analysis
All values are expressed as means ± Standard Deviation (St. Dev.) Student's t-test is used for statistical analysis of Table 2 and ANOVA test was used for statistical analysis for Table 1 and a confidence level of p < 0.05 was chosen as an indication of statistical difference.

Conclusions
SD suppresses multiplication of melanoma B 16 F 10 cells, diminishes permeability for Ca 2+ ions and decreases protein levels of COX-2, and increases degradation of phospholipases PLA 2 and PLC γ 1 and diminishes enzymatic activity of the Ca 2+ -dependent cPLA 2 . These finding supports our previous data suggesting that sarcophine-diol may have a significant potential in treatment of melanoma.