Induction of apoptosis and cell cycle arrest by Negombata magnifica sponge in hepatocellular carcinoma.
Hanaa M Rady 1(✉) . Amal Z Hassan 1 . Sohair M Salem 2 . Tahia K Mohamed 1 . Nora N Esmaiel 2 . Mohamed A EzEl-Arab 3 . Mohamed A Ibrahim 1 . Fayez K Fouda 4 .
National Research Centre-Egypt. Chemistry of Natural compound Department1, Molecular Genetics Department 2, The National Institute of Oceanography and Fisheries (NIOF) 3, National Research CentreEgypt. Hormones Department4. *corresponding to: Hanaa Mahrous Rady (Tel. +20201026096060, Fax +20237492816, E-mail:
[email protected]). Address: National Research Center. El-Buhouth St., Dokki, Cairo, Egypt. Postal code: 12622.
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ABSTRACT: Marine sponges have been considered as a gold mine, with respect to the diversity of their secondary metabolites. Many sponge extracts and isolated compounds are potential anticancer agents. In the present study, the antiproliferative activity of Negombata magnifica was investigated as petroleum ether extract (PE), total methanolic extract (ME) and two sub-fractions II and III, isolated pure compounds (palmitic acid and pregnanediol), sponge mesohyl and primmorphs ethyl acetate extract. Palmitic acid was used as a positive control. Cell viability was assessed via MTT assay. Apoptosis was investigated in terms of DNA fragmentation and BCl2 gene expression. Cell cycle analysis was performed via flow cytometry. GLC analysis of petroleum ether extract (PE) revealed that it contains 84.46% hydrocarbons, 15.54% sterols whereas, the fatty acids contain 38.62 % saturated and 61.38 % unsaturated fatty acids. Results revealed that except for pregnanediol and fraction II, all treatments exhibited cytotoxic activity. Primmorphs ethyl acetate extract, and fraction III arrested cells in G0-G1, while fraction II arrested cell cycle in G2/M. PE extract arrested cells in G0-G1 and G2/M. Mesohyl, petroleum ether and fraction III could be apoptotic agents as indicated by DNA fragmentation independent on BCL2 expression, while ME and pregnanediol could exhibited pro-apoptotic effect through decreasing BCL2 expression although no DNA ladder was observed. Key words: Negombata magnifica, mesohyl, primmorphs, antiproliferation, cell cycle, apoptosis.
Introduction Sponges (Porifera), the simplest multicellular organisms, do not have organs or true tissues. However, they have specialized cells that can carry out distinct functions within the organism. This is generally referred to as cellular-level organization (Weissenfels and Stniegler 1979). The body of a sponge consists of two cell layers separated by a gelatinous region, the mesohyl, which is the only layer of the sponge body wall that is not bathed with the environmental water. As a connective tissue, the mesohyl is composed of a proteinaceous, gel-like matrix (mainly galectin, collagen, fibronectin-like molecules, and a minor component, dermatopontin) that contain differentiated and undifferentiated cells as well as skeletal elements (Ax 1996). Sponges produce toxins and other compounds to repel and deter predators, compete for space with other species and for communication and protection against infection (Uriz et al. 1996). Many of these compounds exhibited cytotoxic and anticancer activity (Belarbi et al. 2003, Zhang et al. 2003). This in turn will provide a window of producing active metabolites with high therapeutic value. Based on the fact that mesohyl provide the platform for specific cell adhesion, signal transduction and cell growth (Müller 2003), it could has a role in modulation of cancer cell phenotype. The quantity of extractable sponge biomass available from the sea cannot usually meet the demand for large scale research and clinical development of antitumor compounds. This bottleneck is often referred to as the “supply problem” in the commercial development of sponge-derived drug leads. Among various technologies proposed to solve this problem is the production of the primmorphs which is a culture system capable of DNA synthesis, cell proliferation, and the synthesis of bioactive metabolites (Osinga et al. 1999). Primmorphs are a special form of 3D-cell aggregates from sponge cells, they are smooth spherical aggregates covered with a collagen-like skin layer. They can be used as biofermenters for the production of bioactive secondary 2
metabolites. For example, the successful production of the secondary metabolite avarol was achieved using primmorphs from D. avara (Müller et al. 2000). Some anti-cancer reagents cause cell death through interfering with the processes of cell cycle (Dirsch et al. 2002) and some others cause cell death by apoptosis (Gamet-Payrastre et al. 2000), which plays an important role in the balance between cell replication and cell death. Cancer and many human diseases are cell cycle diseases. Cell cycle coordination takes place mainly at G1/S and G2/M phase transitions by a series of checkpoints. It has been shown that the activities of many regulatory factors of checkpoints are lost or arrested during the process of tumorigenesis (Liu et al. 2004) where some anti-tumor reagents could restore the altered regulatory checkpoints (Agami and Bernards 2000). Cell cycle arrest may ensure that cells have time to repair the damages, whereas apoptosis may eliminate the damaged cells (Deng 2006). Bcl-2 is the first protein identified to protect a cell from undergoing apoptosis (Gross et al. 1999). Our previous study by Rady (2014) was the first highlighted the use of mesohyl, itself, of the four sponge species as antitumor agents. The presented study is the first to produce Negombata magnifica primmorphs using its mesohyl without sponge cell separation. The use of these sponge products (mesohyl and Primmorphs) to produce anticancer agents will facilitate the commercial development of sponge derived therapeutics instead of consuming sponge fortune. In the present study various sponge products of Negombata magnifica including mesohyl, ethyl acetate extract of its primmorphs, petroleum ether extract (PE), crude hydromethanolic extract (ME) and its chromatographic fractions II and III as well as the isolated compounds, palmitic acid and pregnanediol, have been prepared and tested as antiproliferative agents against human liver carcinoma cell line (HepG2).
MATERIALS AND METHODS Sample collection and preparation The red sponge Negombata magnifica (Figure 1) was collected from the Red Sea (Hurghada, Egypt). Sponge specimens were immediately transferred to the laboratory. Healthy fresh sponge specimens were soaked in sterile natural seawater (NSW) supplemented with 25 ppm CuSO 4 for 3h to kill protozoan contaminations and washed three times with sterile NSW to remove CuSO4. The sponge was then soaked in sterile calcium-magnesium free sea water (CMFSW) for 1h in antibiotic mixture (streptomycine-penicillin-garamycin).
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Primmorphs production and extraction Under sterile condition, in a laminar flow, sponge specimen was dissected. The mesohyl was aspired carefully and collected with a sterile syringe; care should be taken not to aspirate any other fluids than the mesohyl. For primmorphs production, under sterile conditions, fresh mesohyl was diluted 1:2 (total cell density 30 X 107 cells/ml) using sterile natural sea water, then incubated at 16 ºC with discontinuous gentle agitation. Primmorphs were counted and weighed then extracted by ethyl acetate. The solvent was distilled off under reduced pressure at 40ºC then the extract was dissolved in dimethyl sulfoxide (DMSO) then diluted with RPMI media for cell culture and prepared to the bioassays. The ethyl acetate extract of primmorphs was subjected to GC/MS to identify its chemical composition. Bioactive materials extraction and isolation General Experimental Procedures NMR spectra were acquired on a Bruker AV NMR spectrometer (Bruker Biospin, Bruker Inc.) operating at 400 (1H) and 100 MHz (13C). The GC/MS analysis was performed using a Thermo Scientific, Trace GC Ultra / ISQ Single Quadrupole MS, TG-5MS fused silica capillary column (30m, 0.251mm, 0.1 mm film thickness). The identification of the compounds was performed based on the comparison of their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC/MS system. GLC conditions of the unsaponifiable matter and fatty acids was performed on Hewlett Packard-HP 6890 series, GC system, equipped by flame ionization detector. The fresh, cleaned sample of N. magnifica (400 g) was cut into small pieces 1cm each, grounded and extracted successively using petroleum ether and 80% methanol. Each extract was evaporated till dryness under vacuum. Petroleum ether extract (PE) was subjected to saponification according to the method reported by Tsuda et al. (1960). The identification of the fatty acids were achieved through their methyl esters using GLC (Sheppard and Iverson 1975). The crude methanol extract (ME) 2.80 g was subjected to silica gel column and eluted with n-hexane / DCM / MeOH gradient. Based on the TLC monitoring, three fractions were obtained; I (0.65 g), II (0.64 g) and III (0.70 g). Fraction I was subjected to GC-MS analysis while, fraction II was chromatographed on silica gel using DCM / MeOH gradient and after monitoring by TLC, the fractions (9.5: 0.5) were combined and subjected to further purification using Sephadex LH-20 (DCM / MeOH, 60:40) to afford 30 mg of pure compound 1. Fraction III was dissolved in (400 ml) MeOH whereas, a white precipitate
deposited. This was filtered and recrystallized from DCM / MeOH to afford 50 mg of pure compound 2. Fatty acid methyl ester of compound 2 was analyzed by GLC. Cell line propagation Human tumor liver cell line, hepatocellular carcinoma (HepG2) was supplied by Naval American Research Unit–Egypt (NAmRU). Cells were propagated and maintained in RPMI-1640 medium with L-glutamine (Sigma) and supplemented with 10% fetal calf serum (Sigma) for growth and 2% for maintenance medium, and 1% antibiotic mixture (20 units/ml of penicillin G sodium and 20 mg/ml streptomycin sulfate, Gibco).
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Treatment of tumor cells The cells at approximately 80% confluence were selected for trypsinization. The cell suspension in RPMI-1640 culture medium were prepared, seeded and incubated at 37°C and 5% CO2 overnight. Cells were treated with different concentrations of the treatments. MTT assay Cytotoxicity against HepG2 cells was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide] assay )Mosmann 1983). Briefly, cells were seeded in 96-well microplates (3 X 103 cells / well) in 100 µl RPMI-1640 culture medium and incubated at 37 °C and 5 % CO2 overnight. The cells were treated and re-incubated for 48 h. MTT (0.5 mg/ml) solution was added to each well (100 µl) and the cells were incubated over night until the purple formazan crystals appeared. The medium was discarded; 100µl of DMSO was added to dissolve the crystals. The optical density (OD) of solubilized formazan was measured at 570 nm using an automatic microplate reader. Results are expressed as percent of control. DNA fragmentation DNA isolation was done according to the manufacturer's instructions (Qiagen) (Xue et al. 2012). Briefly, 5 x 106 cells were collected, washed with PBS buffer, and lysed in 500 μl lysis buffer for 10 min. Following the addition of 25 μl proteinase K, the lysate was incubated at 50°C for 1 h, centrifuged, and washed. Genomic DNA was eluted with 30 μl elution buffer. DNA samples were loaded onto a 2 % agarose gel containing 0.1 mg/mL ethidium bromide, electrophoresed and visualized by UV light. Real-time Quantitative RT-PCR Total RNA was isolated using Biozol. RNA was reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (thermo scientific) according to manufacturer. For quantitative Real-Time PCR, amplification mixtures was prepared using KAPA SYBR®FAST q PCR Kit (KAPA BIOSYSTEM). Beta actin was used as an internal reference gene to normalize the expression of BCL2. The results were expressed as the ratio of reference gene mRNA to target gene mRNA using 2-ΔΔCt method. The primers sequences of Bcl2 and β-actin were previously reported (Yang and Chan 2009, Borth et al. 2011) Cell cycle analysis HepG2 cells were incubated at 5 × 105 cells/well in 6-well plates with RPMI1640 medium for 12 h, then treated with different sponge materials for 48 h. Cells were harvested and fixed in 70% ice-cold ethanol at -20°C overnight. After fixation, cells were washed with PBS, resuspended in 1 mL PBS containing 1 mg/mL RNase (Sigma) and 50 μg/mL PI (Sigma), and incubated at 37°C for 30 min in the dark. Samples were then analyzed for DNA content by flow cytometry (Beckman, USA), and cell cycle phase distributions were analyzed with the Cell Quest acquisition software (BD Biosciences).
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Statistics Statistical evaluation of the results was done using SPSS version 11. Data were expressed as mean ± SD of percentage of control. One-way analysis of variance (ANOVA) was used to assess significant differences which were considered significant at P< 0.01. Results and Discussion Marine sponge contains some metabolites which can be used in defense, secreted into the boundary layer of the sponge for external defense-offense or released into the mesohyl for internal defense (Zea et al. 1999). The mesohyl is the only layer of sponge body wall that typically is not bathed with environmental water. In this sense mesohyl is the sole internal compartment of the body. These bioactive metabolites in addition to sponge cells, which abundant in mesohyl, exhibits a wide range of biological activities (Frederick and Harrison 1991). There is evidence that some compounds originally found in sponge cells are synthesized by microorganisms associated with sponges. Approximately 40 to 60% of sponge biomass consist of microorganisms, which are located mostly extracellular in the mesohyl (McClintock and Baker 2010). On the other hand, the technique of sponge cell culture, especially the primmorph system, is now available for biotechnological applications. It could be reported that the primmorphs produce similar levels of the bioactive compounds as sponges in the field. In future an up-scaling from the laboratory size, hitherto used, is required to allow larger production of bioactive compounds from sponge primmorphs in bioreactors (Müller et al. 2000). All these information suggested that sponge mesohyl as well as primmorph contain system of a group of chemical compounds that capable to fix the defects in proliferation process which is the main problem in cancer cells. In the present work we measured the antiproliferative effect of the mesohyl of Negombata magnifica and ethyl acetate extract of its primmorphs, petroleum ether extract, the total ME extract and its chromatographic fractions II and III, as well as the isolated compounds 1 and 2 against hepatoma cells (HepG2) in vitro. Cell viability was evaluated using MTT. Apoptotic activity was assessed by DNA fragmentation and Bcl2 expression. Cell cycle phase distribution was investigated by flow cytometry using propidium iodide (PI).
Chemical characterization The identification of fatty acids, sterols and hydrocarbons of PE extract of N. magnifica was achieved by comparing the retention time of their peaks in GLC with those of authentics (Table 1). PE extract was found to contain 38.63 % of saturated fatty acids and 61.37 % of unsaturated fatty acids. Palmitic acid (24.63 % of the saturated fatty acids) and linoleic acid (26.43 % of unsaturated fatty acids), were the major constituents. GLC analysis of the unsaponifiable fraction of the PE extract revealed that hydrocarbon compounds was the major content (84.46 %) in which eicosane is the major compound with the presence of nonadecane, tricosane and octadecane. Sterols represented 15.54 % where, they are composed mainly of cholesterol, campasterol, stigma sterol and β- sitosterol. The crude ME extract was subjected to silica gel column with n-hexane / DCM / MeOH gradient elution to get three fractions; I, II and III The fraction I was subjected to GC-MS analysis showing four known components which were assigned as canthaxanthin (RT: 12.00, 1.98 %), 1,2 7
propadiene (RT: 13.49, 11.63%), p- menthane 1,8- diol (RT: 21.09, 12%) and stigmast-5-en-3-ol (RT: 55.26, 74.39%). Fraction II was chromatographed on silica gel using DCM / MeOH gradientand after monitoring by TLC, the fractions (9.5: 0.5) were combined and subjected to further purification using Sephadex LH-20 (DCM / MeOH, 60:40) to afford 30 mg of pure compound 1 (pregnanediol). Fraction III was dissolved in (400 ml) MeOH whereas, a white precipitate deposited. This was filtered and recrystallized from DCM / MeOH to afford 50 mg of pure compound 2. Compound 1: White solid; Rf=0.41, 5% DCM / MeOH; m.p:130°, Comparison of its structural analysis using 1 H, 13 C NMR and MS with the previously published data (Blunt and Stothers 1977) confirmed the identity of compound 1 as pregnandiol (Figure 2). Compound 2: White precipitate; Rf= 0.58, 10% hexane / DCM; crystallized from DCM / MeOH; m.p.61-63°. GLC analysis of the prepared methyl ester of compound 2 was carried out and by comparing the relative retention time of the peak with those of the pure available authentic standards. This compound was identified as palmitic acid (Figure 2). The structure elucidation (1H and 13C NMR) of compound 2 was found to be in complete agreement with the proposed structure for palmitic acid (Joshi et al. 2009).
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Table 1: GLC analysis of the lipoidal matter of Negombata magnifica Fatty Acids
Sterols and Hydrocarbons RT 18.63 19.13 21.12 24.88 33.52 34.52 35.21 37.23
Compound Octadecane Nonadecane Eicosane Tricosane Cholesterol Campasterol Stigmasterol β - Sitosterol
% 1.96 16.99 53.95 11.56 9.29 1.39 2.27 2.59
RT 19.22 20.49 20.62 20.74 21.43 29.17
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Compound Palmitic acid (16:0) Stearic acid (18:0) Oleic Acid (18:1) Linoleic Acid (18:2) Linolenic Acid (18:3) Behenic Acid (22:0 )
% 24.63 9.00 14.56 26.43 20.38 5.00
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Primmorphs production Fresh mesohyl was diluted 1:2 (total cell density 30 X 107 cells/ml) using sterile natural sea water or modified artificial sea water prepared according to Zhao' basal medium (Zhao et al. 2008) at 16ºC with discontinuous gentle agitation resulted in the production of 1-2 mm red smooth round primmorphs formed in suspension within 3-5 days as observed by inspection with inverted light microscope (Figure 3), at the 5-8th day the primmorphs can be examined with naked eye. Primmorphs were collected, counted then weighed. Unfortunately, continuous subculture of primmorphs under our lab conditions failed but further trails will be achieved. Transmission electron microscope of the primmorph was represented in figure 4.
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Cytotoxicity Except in case of pregnanediol and fraction II, cytotoxic activity was induced by all other treatments against HepG2 cells. IC50 values of mesohyl, ethyl acetate extract of primmorphs, PE, ME, fraction III, and palmitic acid (positive control) were 5µl /ml, 3 µg/ml, 5 µg /ml, 10 µg/ml, 10 µg /ml and 0.5 µg /ml, respectively. All the treatments used in this study were less toxic than the positive control, palmitic acid. Mesohyl exhibited a cytotoxic activity against HepG2 cells, although it considered a good natural medium for living sponge cells which have high telomerase activity (Custodio et al. 1998). The cytotoxic activity of PE extract could be attributed to its hydrocarbon, sterol contents (Hashem et al. 2014) and the high percentage (61.38%) unsaturated fatty acids which have been reported to exhibit cytotoxicity against cancer cells (Hayashi et al. 1998) especially linoleic acid which has significant anticancer effects (Trimborn et al. 2000, Achenef and Arifah 2012). The cytotoxic activity of fraction III could be attributed to the presence of palmitic acid (a major component) which considered as a lead compound of anticancer drugs (Harada et al. 2002).
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Induction of Apoptosis Bcl2- gene expression and DNA ladder Considering two fold changes as a cut off value, only ME extract and pregnanediol affected Bcl2 expression by decreasing it 3.3, and 16 fold respectively, however they had no effect on DNA ladder. This suggest that they can cause a release of pro-apoptotic proteins (Del Gaizo Moore et al. 2007). On the other hand, mesohyl, fraction III and PE exhibited DNA ladder with no effect on Bcl2 expression. This may suggest that they can induce apoptosis independent on Bcl2. The decreased BCL-2 expression observed in case of the treatment with ME extract may be attributed to the presence of isolated pregnanediol and cytotoxic palmitic acid which known to induce apoptosis. The cytotoxic primmorphs extract and non-cytotoxic fraction II did not change neither BCL2 gene expression nor DNA ladder. Palmitic acid (positive control) induced apoptosis by decreasing BCL-2 expression 4.3 fold and the induction of DNA ladder (Dyntar et al. 2001). Data were summarized in figure 6 and Table 2.
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Cell cycle analysis Based on cell cycle analysis, red primmorph ethyl acetate extract, PE, fractions II and III decreased cell population in S-phase to 7%, 2%, 17.8% and 9%, respectively. Cells were arrested in G0-G1 (84.5%, 85% and 84%) in case of treatment with red primmorph ethyl acetate extract, PE, and fraction III, respectively. While PE and fractions II arrested cell populations in G2/M (11% and 8.9% respectively). Cell cycle arrest in G0-G1 and G2-M may cause slow growth and allow a damaged cell to undergo apoptosis induce cell death. Mesohyl and prgnanediol did not exhibit any effect on cell cycle phases. All treatments have no effect on G2/G1. Data were represented in figure 7 and Table 2. Palmitic acid (positive control) decreased cell population in S-phase to 14%, arrest cells in G0-G1 (Artwohl et al. 2004) in a percent = 83%.
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Table 2: Summary of the results, cytotoxicity, cell cycle arrest and apoptotic induction induced by Negombata magnifica.
Cell cycle
Apoptosis
Treatment
Cytotoxicity (IC50)
S-phase
G0-G1
G2/M
Control
0
21.0%
72.5%
4.5%
-
-
Mesohyl
5 µl/mL
21.0%
71.0%
6.5%
2
+
Primmorph Extract Petroleum Ether Extract (PE) Methanolic extract (ME) Palmitic Acid
3 µg/mL
7.0%
84.5%
6.5%
0.87
-
5 µg/mL
2.0%
85.0%
11.0%
0.5
+
10 µg/mL
18.0%
73.6%
6.5%
0.3
-
0.5 µg/mL
14.0%
83.0%
1.1%
0.23
+
Pregnanediol Fraction II
Non cytotoxic Non cytotoxic
21.0% 17.8%
75.0% 73.2%
2.0% 8.9%
0.06 0.5
-
Fraction III
10 µg/mL
9.0%
84.0%
5.0%
2
+
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BCL2 expression DNA Ladder (fold change)
Conclusion PE extract and fraction III are the most promising agents where they exhibited cytotoxic effect, BCl2 independent apoptotic induction (DNA ladder). In addition, they decreased cell population in S-phase and arrested cells in G0-G1 and G2/M. Primmorphs extract has a cytotoxic activity without inducing apoptosis. It had a pronounced cell cycle effect where it decreased cell population in S- phase and arrested cells in G0-G1. Mesohyl exhibited apoptotic activity as indicated by DNA ladder. The most significant effect on Bcl2 expression is indicated in pregnanediol which means it can induce apoptosis without having cytotoxic activity (Essack et al. 2011). No obvious antiproliferative activity was observed in case of treatment with fraction II.
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