Antiproliferative activity of protein extracts from the black clam ( Chione fluctifraga ) on human cervical and breast cancer cell lines

1 Posgrado en Biociencias, Departamento de Investigaciones Científicas y Tecnológicas (DICTUS), Universidad de Sonora, Hermosillo, Sonora, 83000, México. 2 Departamento de Física, Universidad de Sonora, Hermosillo, Sonora, 83000, México. 3 Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Sonora, 83000, México. 4 CTAOA, Centro de Investigaciones en Alimentación y Desarrollo A.C, Carretera a la Victoria Km 0.6, Hermosillo, Sonora, 83304, México.


INTRODUCTION
Cancer is a worldwide health issue; it is not just one disease but a variety of different diseases, which have in common uncontrolled cell proliferation.There are more than 100 different types of cancer, most of them take the name of the organ or cell in which they begin to grow (Hawkins, 1992;Martinez et al., 2003).Breast and cervical cancer are the main cancer related cause of death in women around the world.The high prevalence of these types of cancer is due to a lack of strategies that allow detection at early cancer stages (Palacio-Mejía et al., 2009;Gök et al., 2011).Chemotherapy is the most commonly used treatment against cancer, however some patients develop resistance against the drugs and this has become a major problem in the past years.Resistance to treatment can be caused by a large variety of factors, which include individual variations between patients and genetic differences among tumor cells (Gottesman, 2002;Jordan and Wilson, 2004;Bhutia and Maiti, 2008).The discovery of new compounds from natural sources has been very important in pharmaceutical science research.The past decade has seen an increase in the number of compounds obtained from the screening of diverse marine invertebrates, such as soft corals, tunicates and mollusks, that are advancing to preclinical trials (Liang et al., 2008;Simmons et al., 2005;Amador, 2003;Bhatnagar and Kim, 2010).Examples of anticancer agents extracted from marine organisms include bryostatin-1, a macrocyclic lactone from Bugula neritine, with antitumor activity; ziconotide, a peptide from cone snail, is an N-type calcium-channel blocker in chronic pain treatment; ectenaisdin 743 is a tetrahydroisoquinolone alkaloid, isolated from Ectinascidia turbinate, with cytotoxic activity.It has been previously reported that the peptide dolastatin, obtained from the marine mollusk Dollabella auricularia, possesses an impressive growth inhibition of cancer cells.Another example is Kahalalide F, a cyclic depsipeptide obtained from Elysia rufescens; this peptide shows antitumor activity against colorectal, lung and prostate cancer cells, being lysosomes the cellular target (Haefner, 2003;Amador, 2003;Aneiros and Garateix, 2004;Faircloth and Cuevas, 2006;Bhatnagar and Kim, 2010).
Many peptides with different biological activities such as antioxidant (Wenyan et al., 2012), immune-regulatory (Lixin et al., 2005), antihyperglycemia and antihyperlipemia activity (Tsai et al., 2008) have been isolated from the clam Meretrix meretrix; polypeptide Mer2, MGP 0501 and MGP 0405 inhibits cell proliferation of several cancer cell lines; Mere-15 shows antitumor activity in vitro and in vivo; the protein (MML) shows growth inhibition in several cancer cell lines such as breast, colon and hepatic (Leng et al., 2005;Ning et al., 2009;Wang et al., 2012).Two peptides, 18 and 16 kDa, with cytotoxic activity in vitro against HeLa cells have been purified from Arca subcrenata (Song et al., 2008).The Gulf of California has a great diversity of clams, such as the black clam (Chione fluctifraga), that represent a popular seafood and a very important income source for the community (Martinez-Cordova and Martinez-Porchas, 2006).The aim of the present study was to evaluate the capability of C. fluctifraga's protein extract to inhibit the cellular growth on HeLa and MDA-MB-231 cultured cells.

Materials
The clams (C.fluctifraga) were obtained from the experimental unit of the University of Sonora (Kino Bay, Sonora).All the reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of the crude extract
The clams were cut into small pieces and homogenized with 0.01 M phosphate buffer (0.1 M NaCl, pH 7.2) in a 1:3 ratio (w/v).The homogenate was centrifuged at 3000 x g.The supernatant was fractionated by ammonium sulfate precipitation up to 60% saturation.After 12 h of stirring the precipitate was collected by centrifugation (3000 x g, 10 min) and the pellet was suspended in phosphate buffer and collected as the crude extract.

Preparation of protein extract
The crude extract was passed through a Sephadex G-25 column (1.5 × 50 cm) pre-equilibrated with 0.01 M phosphate buffer (0.1 M NaCl, pH 7.2) at a flow rate of 1 ml/min and the absorbance was monitored at 280 nm.The fraction collected was dialyzed against 0.025 M Tris-HCl and then loaded onto a HiPrep 16/10 Q XL column (BioRad) pre-equilibrated with 0.025 M Tris-HCl pH 8.2.The column was eluted with a linear gradient 0 to 1 M NaCl prepared in Tris-HCl buffer at a flow rate of 1 ml/min, each fraction was collected at a volume of 5 ml and detection was performed at 280 nm.Fractions were lyophilized then dialyzed and antiproliferative activities were determined.

Measurement of protein content
The protein concentration was measured by the BCA method using the BCA protein assay kit (Pierce, USA) and bovine serum albumin as standard.

Tricine-SDS-PAGE
The extraction process was monitored by electrophoresis tricine-SDS-PAGE (Schägger, 2006) using a Bio-Rad Mini-Protean II electrophoresis unit (Bio-Rad, CA, USA) with an acrylamide concentration of 16% for the running gel.The protein bands were detected by silver stain.

Statistical analysis
The experimental data were expressed as the mean ± standard deviation.Statistical analysis was carried out using the GraphPad PRISM 6 program (GraphPad Software, Inc., San Diego, CA), statistical significance (p < 0.05) was determined using a Student´s t-test.

RESULTS AND DISCUSSION
Marine organisms represent a large source of new compounds with biological activities.Direct extraction is one way to obtain bioactive compounds from marine organisms; this approach is widely used for isolated and purified biologically active peptides (Chen et al., 2013).Figure 1 shows the crude extract's chromatogram.Three fractions were obtained after passing the crude extract through the Sephadex column.The separation is given based on the size, the high molecular weight proteins were obtained in the first fraction and afterwards the low molecular weight ones.The fractions that resulted (F1, F2, and F3) from gel filtration chromatography were separated by ion-exchange chromatography through elution with a NaCl gradient on a HiPrep 16/10 QXL column, ten fractions were obtained as follows: four fractions from F1, three fractions from F2 and three fractions from F3 (Figure 2).Antiproliferative activity of all fractions was tested using the MTT assay, a wellestablished in vitro model for citotoxicity against cancer cell lines that has been used as a conventional technique for the screening of new compounds with antiproliferative activity (Song et al., 2008).F1 showed a antiproliferative effect of 87% of over MDA-MB-231 cell and 43.17% over HeLa cell.F3 shows inhibition of the cellular growth of 64.36 and 70% on HeLa and MDA-MB-231 cell, respectively (Figure 3).When a high concentration of the extract is required to inhibit the cellular growth on 50% of the population, it is considered a low cytotoxicity (Hsu et al., 2010).The concentrations used ranged from 0.054 to 1.97 mg/ml, inhibition on cancer cell line has been reported with 1 mg/ml of extracts (Picot et al., 2006;Liang et al., 2008).The fractions did not inhibit cellular proliferation beyond 60% on the human retinal pigment epithelial cells (ARPE-19).Ten fractions were obtained by ion-exchange chromatography, on the antiproliferative evaluation of these samples (Table 1)  reported IC 50 values of 84.9 and 215.3 µg/ml over A549 and CCRF-CEM cells, for Syngnathus acus's protein purified by ion-exchange cromatography; from A. subcrenata two fractions have shown cytotoxic activity against HL-60 (IC 50 : 123.2 µg/ml) and HeLa cells (IC 50 : 38.2 µg/ml) (Song et al., 2008).
Several biologically active peptides from marine organisms have been identified and have shown different activities including antioxidant, antimicrobial, antiproliferative and antihypertensive actions (Kim et al., 2013).Some of these compounds are in the process of entering clinicals trials.Peptides from marine sources (Jaspamide, aplidine, didemnin, MML, Mere 15 and dolastatin) induce cell death with different mechanisms, including apoptosis, disruption of microtubular function; interaction with tubulin, alteration of microtubule assembly, or angiogenesis inhibition (Bai et al., 1990;Zheng et al., 2011).Also some of these compunds can bind the vinca alkaloids or the colchine-binding domain inhibiting the polimerization of microtubules (Simmons et al., 2005;Amador, 2003).Several bands with molecular weights between 45 and 18 kDa were observed in the electrophoresis gel (Figure 4).Several compounds derived from marine organisms that have been evaluated on pre-clinical and clinical trials as potential anticancer drug are low molecular weight compounds (Ning et al., 2009).
Cyplisin, a 56 kDa protein isolated from Aplysia punctate, has shown citotoxicity against several cancer cell lines; MML, a 40 kDa protein from M. meretrix, has inhibitory effects on the proliferation of human hepatoma,   apparently increasing the permeability of the cell membrane and inhibiting tubulin polymerization.Also, peptides with antiproliferative activity, such as MGP 0405 and MGP 0501 (9.6 and 15.8 kDa, respectively), have been reported (Hoarau et al., 2002;Lixin et al., 2005;Nakano et al., 2006;Takahashi et al., 2008;Zheng et al., 2011;Wang et al., 2012).Takahashi et al. reported a protein with antimicrobial activity of 58 kDa from clam (Ruditapes philippinarum) and Hoarau found fractions of 20 to 28 kDa with detoxify activity.It has been reported that bioactive peptides or small proteins are highly influenced by their molecular weight and structure, which are affected by the isolation process; different approaches for isolation can lead to different bioactive agents (Guo-fang et al., 2011;Wang et al., 2012).
In the present study, the in vitro antiproliferative activity of protein extracts from C. fluctifraga was evaluated for the first time, the importance of these results is evidenced by previous reports of many anticancer chemical entities found by the screening of natural sources, such as marine organisms.Our results suggest that protein extracts from C. fluctifraga could induce cellular growth inhibition against HeLa and MDA-MB-231 cells, however further studies are needed.

Figure 1 .
Figure 1.Fractionation of crude extract by gel filtration chromatography on a Sephadex G-25 column.Column specification: 1.5 x 50 cm; the elution was performed with 0.1M phosphate buffer at a flow rate of 1 ml/min.The elution was monitored at 280 nm.
, the fraction 3F3 showed to inhibit the proliferation of HeLa and MDA-MB-231 cell with IC 50 values of 138.035 and 157.19 µg/ml respectively and 2F2 fraction showed IC 50 values of 67.46 µg/ml over HeLa cells.Wang et al. (2012) have 0.08

Figure 2 .
Figure 2. Fractionation by anion exchange chromatography on a HiPrep 16/10 QXL column.The elution was performed with a linear gradient of NaCl (0-1 M) in 25 mM Tris-HCl buffer at a flow rate of 1ml/min.The elution was monitored at 280 nm.F1: Fraction 1 from gel filtration chromatography; F2: Fraction 2 from gel filtration chromatography and F3: Fraction 1 from gel filtration chromatography.

Figure 3 .
Figure 3.The inhibitory effect of fractions obtained by gel filtration chromatography.The cancer cell lines were treated with the indicated concentrattion of protein and cell proliferation was determinated by MTT assay.A: Fraction 1; B: Fraction 2; C: Fraction 3.

Table 1 .
Antiproliferative activity of fractions obtained by anion exchange chromatography against different cell lines (IC50 µg/ml ± SD).