Antiproliferative effect of the Red Sea cone snail, Conus geographus

Purpose: To investigate the antiproliferative effect of the Red Sea cone snail, Conus geographus, against 4 MCF-7 (breast), MDA-MB-231 (epithelial human breast), HepG2 (hepatocellular) and SKOV-3 (ovarian) cancer cell lines. Methods: Extraction of Red Sea cone snail sample with a mixture of CH2Cl2 and CH3OH (1:1, v/v) yielded 0.55 g of a green viscous material. The cytotoxic effects of the organic extract against the cancer cell lines were determined using cell proliferation (MTT) assay, and the half-maximal concentration (IC50) values measured. The effect of the crude extract on the cell cycle of the HepG-2 was determined by flow cytometry. Results: The extract produced significant inhibitory effects against SKOV-3, MDA-MB-231, MCF-7 and HepG2, with IC50 values of 22.7 ± 2.2, 68.7 ± 6.2, 47 ± 4.2 and 19 ± 2.1 μg/mL, respectively. Cell cycle analysis revealed that the extract enhanced accumulation of HepG2 cells in the Go/G1 phase, at a level of 23.4 and 24.1 % at IC50 (19 μg/mL) and 1⁄2 IC50 (9.5 μg/mL), respectively, when compared to the untreated cells. Conclusion: These results indicate that C. geographus extract exhibits potent cytotoxic effect against HepG2 cells via a mechanism involving G0/G1 cell cycle arrest. Thus, C. geographus is a potential source of a new anti-cancer agent.


INTRODUCTION
High biological diversity and harsh environmental conditions are the main features of marine ecosystems, and they reflect different archetypes of natural compounds, when compared to those originating from terrestrial organisms. Indeed, several new compounds of marine origin are discovered every year [1,2]. Many of these natural products have already been considered in clinical trials with regard to their antitumor activities. Marine organisms offer many challenges and great opportunities for drug discovery, particularly anticancer agents [3][4][5][6].
Conus is a widely distributed genus in tropical and sub-tropical areas. It includes more than eight hundred identified species, which are characterized by their specialized feeding behavior in which they overcome their prey by injection of potent neurotoxic and paralytic venoms [7].
The present study was carried out to investigate the cytotoxicity of the cone snail C. geographus from Saudi territorial waters against SKOV-3, MCF-7, MDA-MB-231 and HepG2 cancer cell lines.

EXPERIMENTAL Materials
Conus geographus was collected from Rabigh area (21° 29′ 31″ N 39° 11′ 24″ E), Saudi Arabia. A voucher specimen was kept in the Faculty of Marine Science King Abdulaziz University, Saudi Arabia.

Determination of antiproliferative effect of extract
The cancer cells were seeded in 96-well plate at a density of 5000 cells/well and incubated for 24 h at 37°C and 5% CO2. Thereafter, the cells were treated with serial dilution of the Conus extract (50, 25, 12.5, 6.25, 3.125, or 1.56 µg/mL). After 48 h, the viability of each cancer cell line was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT, 5 mg/mL) which measures the activity of mitochondrial succinate dehydrogenase in viable cells [9]. Following 4-h incubation, the resultant formazan crystals were solubilized using 10 % SDS/PBS/0.01 N HCl. After 14 h, the absorbance of the formazan solution was read at wavelengths of 570 nm and 630 nm in a BioTek plate reader (EL x 808, BioTek Instruments, Inc., Winooski, VT, USA). The experiment was performed three times and the standard deviation (SD) was calculated. The IC50 was calculated in terms of the concentration that causes 50 % inhibition of cell growth.

Analysis of cell cycle distribution
HepG2 cells were treated with 9.5 and 19 µg/mL Conus extract, or with 0.5% DMSO as negative control. After 48 h of incubation, the cells were washed twice with 1 x PBS (17-516F, Lonza Verviers SPRL, Belgium) and trypsinized [17-161E, Trypsin-Versene (EDTA), Lonza Verviers SPRL, Belgium]. The detached cells were centrifuged at 2500 rpm for 10 min, and the cells were fixed with 70% ice-cold ethanol for 2 h at -20 °C. The fixed cells were washed with 1 x PBS and centrifuged at 5000 rpm for 15 min. The effect of the treatment on the cell cycle was evaluated by staining the cells with propidium iodide (P1304MP, Invitrogen™) for 15 min in the dark. Fluorescence was measured using BD Accuri™ C6 Plus flow cytometer [9].

Statistical analysis
All statistical analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, La Jolla, CA). Graphs were plotted using GraphPad Prism software, version 6.00 (GraphPad Software, La Jolla, CA).

RESULTS
The results indicated that the C. geographus extract showed good antiproliferative activity against SKOV-3, MCF-7, MDA-MB-231 and HepG2 cell lines. The antiproliferative effect of the extract was compared with that of cisplatin (positive control). Both showed significant antiproliferative effects against SKOV-3 cells, at IC50 values 22.7 ± 2.2 and 16.6 ± 3.0 µg/mL, respectively. They also exerted antiproliferative effects against MDA-MB-231, with IC50 values 68.7 ± 6.2 and 7.3 ± 1.0 µg/mL, respectively. The IC50 values for the antiproliferative effects of extract and cisplatin against MCF-7 cells were 47 ± 4.2 and 22.9 ± 1.87, respectively, while their IC50 values for cytotoxicity against HepG2 were 19 ± 2.1 and 5.5 ± 0.35 µg/mL, respectively. Figure 1 shows the percentage of cell viability after treatment with different concentrations of Conus extract. The mechanism of cytotoxic effect of the extract against HepG2 cells was evaluated by assessing its effect on cell cycle using DNA flow cytometry technique, relative to untreated cells. The cell cycle results are shown in Figure 2 and Table 1, with values estimated as percent of cell viability after treatment of HepG2 cells with the obtained IC50 (19 µg/mL) and ½ IC50 (9.5 µg/mL). .

DISCUSSION
The marine environment enhances the production of a vast array of natural metabolites with diverse molecular structures. Currently, more than 28,600 organic compounds of marine origin have been reported. Marine bioprospection is beneficial and rewarding in the area of cancer therapy. The development of anticancer agents is a reflection of the tremendous impact of natural organisms on the chemotherapeutic drug arsenal, with 49% of anticancer agents approved prior to 2014 being classified either as natural products or products derived directly. Cone snails are marine mollusks belonging to the family of Conidae which has 152 genera. The genus Conus, known as specialized predators, currently has more than 800 recognized species [10][11][12]. They sting humans with different adverse effects. Conus species produce different neurotoxic peptides in their injected venoms. The severity of these hazardous peptides in humans has been reported [13]. Most of these proteinoid substances exhibit effects through the nervous system [10,12]. Conotoxins are important potential drug leads [14,15].
A computer survey based on different scientific data bases (e.g., Scifinder) showed that there are limited publications on the cytotoxicity of Conus geographus against cancer cell lines. Thus, the present study was carried out to investigate the cytotoxic effect of C. geographus against some selected cancer cell lines. The results indicated that the extract of C. geographus increased the population of cells in the Go/G1phase from 72.6 % to 96.0, and 96.7 % at the IC50 value of 19 µg/mL, and ½ IC50 value of 9.5 µg/mL, respectively. At these concentrations, the extract increased the cell population at Go/G1 phase by 23.4 and 24.1 %, respectively. The increase in the population of non-proliferating cells was accompanied by subsequent decrease in cell population in Sphase from 4.8% to zero. Moreover, the population of cells in G2/M phase was decreased from 20.8 % to zero at IC50 and ½ IC50 (19 and 9.5 µg/mL).
It is known that the approach of a cell to the end of the G1 phase is controlled at an important checkpoint called G1/S, where the cell manages no replication of its DNA. At this point, the cell is tested for DNA damage to confirm that it has all the required cellular machinery to allow for effective cell division. Cells with intact DNA continue to S phase, while cells with damaged DNA that cannot be repaired are arrested through apoptosis, or programmed cell death.

CONCLUSION
The results obtained in this study indicate that Conus geographus exerts significant antiproliferative effect against HepG2 through cell cycle arrest at G0/G1 phase. Therefore, C. geographus is a potential source of a new anticancer agent.