Anticancer activities of toxic isolate of Xestospongia testudinaria sponge

Aims: The purposes of this study were to determine the anticancer activity of Xestospongia testudinaria sponge isolate and identify the responsible compounds. Materials and Methods: The metabolites were extracted using methanol maceration at room temperature. The separation and purification of metabolites were performed using fractionation and column chromatography. The toxicity was examined using the brine shrimp lethality assay, and the toxic isolates were tested for anticancer activity against HeLa cells. Gas chromatography-mass spectrometry analysis was used to identify the compounds in the isolate. Results: When the methanol extract was partitioned with n-hexane, chloroform, and n-butanol, the chloroform fraction was the most toxic, with a concentration that results in 50% lethality (LC50) value of 39.81 ppm. After separation of the chloroform extract, fraction B (FB) was the most toxic, with an LC50 value of 44.67 ppm. The isolate from FB showed anticancer activity with a concentration at which 50% of growth was inhibited (IC50) of 2.273 ppm. In total, 21 compounds were identified in anticancer isolates: Nonanedioic acid; tetradecanoic acid; trans-phytol; 2-pentadecanone-6,10,14-trimethyl; pentadecanoic acid; 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-; pentadecanoic acid; 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-; 2,3,7-trimethyloctanal; palmitic acid; docosanoic acid, ethyl ester; 1,E-11,Z-13-octadecatriene; chloromethyl 4-chlorododecanoate; 1-tricosene; 9,12-octadecadienoic acid; 4,8,12,16-tetramethylheptadecan-4-olide; 1-docosene; heneicosane; phosphonic acid, dioctadecyl ester; dodecane,4,6-dimethyl-; n-tetratriacontane; 1-iodohexadecane; and n-heneicosane. Conclusion: These findings indicate that the isolate of X. testudinaria can be used as a natural anticancer toward HeLa cell.


Introduction
Cancer is caused by the abnormal growth and development of cells in the body [1]. During the course of the disease, these cells may spread to other parts of the body, ultimately resulting in death where the currently available therapies are not effective [2][3][4][5]. Cancer is the second most common cause of death after heart disease, responsible for more than 500,000 deaths per year in the United States [6] and an estimated 100 new patients in every 100,000 inhabitants diagnosed every year in Indonesia [7]. Current cancer treatments generally combine surgical and radiation methods with chemotherapy [8], but the methods have not led to optimal results. The development of new anticancer drugs has become a priority because of the high cost and low selectivity of the currently available drugs [9]. A wide range of natural resources, including marine organisms, has been considered in the search for new anticancer compounds [10]. Some researchers believe that sponges are a potential source of bioactive compounds from the ocean. Nearly 5000 compounds have been isolated from these sponges and determined to have various biological effects such as antimicrobial, antifungal, antiviral, and anticancer activities [11]. Metabolite extracts from sea sponges contain bioactive compounds with antiviral [12], anti-HIV, anti-inflammatory, antifungal, antileukemia [13], enzyme inhibitory [14], antimalarial [15], antioxidant [16], cytoprotective, and antitumor [17] activity.
A preliminary test for anticancer activity was conducted using the brine shrimp lethality assay [26]. If material has an LC 50 value of below 1000 ppm, this indicates anticancer potential and suggests the use of further anticancer testing in HeLa cells [19,27]. The methanol extract of X. testudinaria sponge from Sanur Bali had anticancer activity with a concentration at which 50% of growth was inhibited (IC 50 ) of 1327 ppm [19].
The purposes of this study were to determine the anticancer activities of toxic isolates of X. testudinaria sponge from Sanur, Bali, Indonesia, and identify the responsible compounds.

Ethical approval
The study only used invertebrate, so ethical approval was not necessary.

Materials
The X. testudinaria sponge was collected from the coastal waters of Sanur, Bali, on May 9, 2018. Methanol, n-hexane, chloroform, and n-butanol were purchased from Merck, Germany. Brine shrimp Artemia salina eggs were purchased from American Technology. The cell line was purchased from the Primate Study Centre, Bogor Agriculture University. Gas chromatography-mass spectrometry (GC-MS) was performed using a GC-MS-QP2010 Ultra Shimadzu from Japan.

Sample preparation and extraction
Fresh sponge samples were washed with water until clean, cut into small pieces, and dried away from direct sunlight for 7 days. After drying, the sample was sieved through at 100 mesh filter to ensure appropriate homogeneity. In total, 500 g dry powder of sample was extracted using methanol maceration. Every 24 h, the extract was filtered, and the pulp was re-extracted using fresh methanol. This extraction process was conducted 3 times. All the methanol extracts were evaporated using a rotary evaporator to yield the crude extract [19].

Separation and purification
The most toxic extracts were then separated by silica gel column chromatography using suitable eluents to obtain several fractions. All fractions were tested for toxicity. The most toxic fraction was tested for purity by thin-layer chromatography (TLC) using several eluent systems. If the isolate provided a single stain on the TLC plate in various eluent systems, then the isolate was considered pure according to TLC; finally, the anticancer effect of the isolate was determined in HeLa cells [19].

Toxicity test
The medium for larvae hatching was made by filtering seawater. Seawater was placed in an aquarium, which was divided into two parts; one was dark and the other was bright. A. salina eggs (50 mg) were placed or immersed in the dark part and left for 48 h until it hatched into a mature larva and was ready to use for testing. Each methanol and n-EH (20 mg) were dissolved into 2 mL of solvent. These solutions were considered as 500 μL, 50 μL, and 5 μL, respectively; each solution was inserted into the test tube, and the solvent was evaporated. Dimethyl sulfoxide (nearly 50 μL), seawater (1 mL), and 10 larvae were placed into a test tube containing the sample; the solvent was evaporated, and seawater was added to a volume of 5 mL, to obtain extract concentrations of 1000, 100, and 10 ppm. A concentration of 0 ppm (solution without the addition of the extract) was prepared as a control. After 24 h, the death of A. saline larvae was measured. The standard assessment of larval mortality is when the larvae do not show movement during an observation period of several seconds [28]. The number of live and dead larvae was recorded, and the data were analyzed to find the LC 50 .

Anticancer test
The toxic isolate was assayed for its anticancer activity against HeLa cells [29]. HeLa cells were cultured in Roswell Park Memorial Institute 1640 medium, and the initial number of cells was counted using a microscope. The cells were trypsinized, harvested, and centrifuged to form two layers (sediment and supernatant). The supernatant was removed and the precipitate was pelletized; 1 mL of complete medium was added and then the number of cells as counted using a hemocytometer. Subsequently, 2×10 4 cells were seeded in 100 μL Available at www.veterinaryworld.org/Vol.12/September-2019/12.pdf of the medium in a 96-well plate and incubated for 1-2 h to allow the cells to adhere. Subsequently, 100 µL extracts of the test material were added at various concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.91, 1.95, 0.97, 0.48, 0.24, 0.12, and 0.06 μg/mL), to make a total volume of 200 µL in each well. The cells were then incubated for 24 h at 37°C. After 24 h, the cells were observed using the microscope. 3-(4,5-Dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT; 5 μg/mL) was added to each and incubated for 4 h. Subsequently, stop solution of sodium dodecyl sulfate (SDS) 10% in 0.01 N HCl was added into each well and incubated overnight. The absorbance at 500 nm was observed using an enzyme-linked immunosorbent assay plate reader.

Identification of compounds
Anticancer compounds were separated using GC-MS. The mass spectrum of the active isolates obtained was compared with standard reference spectra that were programmed on the device (GC-MS-QP2010 Ultra Shimadzu) [30,31].

Anticancer activity
Anticancer activity against HeLa cells was determined using the MTT assay. The MTT assay is a colorimetric method used to measure cell proliferation. The principle of the assay is the reduction of yellow tetrazolium salt MTT, which is reduced to purple formazan crystals by living mitochondria [32]: MTT is absorbed into live cells and reduced by succinate dehydrogenase in the electron transport chain of mitochondria to formazan. The formed formazan crystals are dissolved in 10% SDS, forming a purple solution. The MTT reduction occurs through the NADH and NADPH cofactor pyridine nucleotides, which only occur in living cells; thus, the amount of formazan formed is proportional to the number of live cells [33]. The optical density (OD) of each well was measured at 595 nm using a microplate reader. All tests were conducted in triplicate, and the average OD value was converted to a percentage inhibition.

Sample preparation and extraction
Fresh sample (10 kg) was washed, cut, and dried for 7 days, which produced 953 g of dried samples. The dried samples were refined to produce 781 g of dry sample powder. Subsequently, 500 g of dry sample powder was extracted using methanol, which yielded 67 g of the methanol extract.

Fractionation
The methanol extract (50 g) was partitioned successively with n-hexane, chloroform, and n-butanol to produce the n-hexane, chloroform, and n-EB, respectively, with yields of 5.17, 3.84, and 9.27 g, respectively. The results of the toxicity test of the three extracts are shown in Table-1. The EC was the most toxic, with an LC 50 value of 39.81 ppm.

Separation and purification
The EC was separated by silica gel column chromatography using n-hexane-chloroform (1.5:8.5) as an eluent, which produced four fractions (Fraction A, Fraction B [FB], Fraction C, and Fraction D). The toxicity data of the four fractions are shown in Table-2. As shown in Table-2, the most toxic fraction was FB, which had an LC 50 value of 44.67 ppm. The FB isolate was then tested for purity using the silica gel TLC method. The tests with various eluent systems all resulted in a single stain, which indicated that the FB isolate was pure, according to TLC.

Anticancer activity
The OD data and percentage inhibition of HeLa cells after treatment with sponge X. testudinaria toxic isolate (FB) are presented in Table-3.
Based on the data in Table-3, the relationship between sample concentration and inhibition was used to determine the IC 50 (Figure-1).
The equation of the graph in Figure-  The IC 50 of the toxic isolate from FB was 2.273 ppm, which is considered a very strong activity [34].

Identification of compounds
The GC analysis of the anticancer is presented in Figure-2. Twenty-one compounds were present in the isolate. The compounds were identified by MS and are presented in Table-4.

Discussion
The data presented in Tables-1 and 2 show that the cytotoxic potency of X. testudinaria sponge extract increased from the EC (39.81 ppm) to FB (44.67 ppm). This indicated that the toxic compounds in the EC exerted a synergistic effect [35]. Based on the anticancer preliminary test (toxicity test), the EC was the most toxic extract, indicating that the toxic compounds are semipolar because they dissolve into chloroform (Table-2) [36]. As shown in Figure-1, the sample concentration was positively correlated with the inhibition of HeLa cell growth. All inhibition percentages of the samples and concentrations were significantly different from the control. Compared with a previous report [19], the anticancer activity of toxic isolates was lower, with an IC 50 of 2273 ppm. This showed that the anticancer compounds in the X. testudinaria sponge exerted synergistic effects.
The anticancer activity (IC 50 = 2.273 ppm) of X. testudinaria sponge toxic isolate agreed with to a previous report [18], in which the IC 50 of ethanol extract of X. testudinaria sponge on HeLa, HepG-2, and Daoy cells was 83.35, 23.45, and 23.31 ppm, respectively. In addition, the n-EH from the same sponge had IC 50 values of 33.7, 30.2, and 20.74 ppm in of on HeLa, HepG-2, and Daoy cells, respectively.

Conclusion
The anticancer activity of X. testudinaria sponge toxic isolates was positively correlated with the inhibition of HeLa cell growth, with an IC 50 of 2.273 ppm. In total, 21 compounds were identified in toxic isolate; three groups of compounds were identified, namely, fatty acids and esters, terpenoids, and hydrocarbons.

Authors' Contributions
MDS designed and conducted the experiment, acquisition of data, and drafting of the manuscript. WSR conducted the experiment and collected the data. NS designed the experiment, drafting of the manuscript. KKA analyzed the data and drafting the manuscript. All authors read and approved the final manuscript.