The effect of ammonium sulfate concentration in protein isolation of lionfish (Pterois volitans) spines venom extract for antitumor test Cite as: AIP Conference Proceedings 2193, 030009 (2019); https://doi.org/10.1063/1.5139346 Published Online: 10 December 2019 Andy Noorsaman Sommeng, Adinda Kemala Eka, Mikael Januardi Ginting, Sonya Pebriani, Muhamad Sahlan, Heri Hermansyah, and Anondho Wijanarko
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AIP Conference Proceedings 2193, 030009 (2019); https://doi.org/10.1063/1.5139346 © 2019 Author(s). 2193, 030009
The Effect of Ammonium Sulfate Concentration in Protein Isolation of Lionfish (Pterois Volitans) Spines Venom Extract for Antitumor Test Andy Noorsaman Sommeng1, Adinda Kemala Eka1, Mikael Januardi Ginting2, Sonya Pebriani1, Muhamad Sahlan1,3, Heri Hermansyah1, Anondho Wijanarko1a 1
Departement of Chemical Engineering, Faculty of Engineering Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia 2
Marine Science Postgraduate Program, Faculty of Mathematics and Natural Science, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia 3
Research Center for Biomedical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia Corresponding author: a)anondho.wijanarko@yahoo.com Abstract. Lionfish (Pterois volitans) is a native species of the Indo-Pacific Ocean. This species is known as destructive invasive species that invade in many areas. The invasion of Lionfish gives negative impacts on the ecology and economic condition of the invaded regions. Therefore, to reduce the number of Lionfish in the ocean and to attract people to exploit Lionfish, the study about Lionfish's benefit is necessary. The recent study revealed that venom extract from Lionfish spines has a potential cytolytic effect that widely uses for antitumor studies. In this study, to obtain the optimum concentration of the solvent that used for protein isolation and to observe the antitumor properties of Lionfish further, we isolated the protein of Lionfish venom extract with ammonium sulfate and tested it on HeLa cell as the model of tumor cells. The Lowry and BSLT test showed that the highest protein concentration and activity obtained when the highest level of ammonium sulfate isolated crude venom. The highest percentage of inhibition in HeLa cells reach 17-19% at the highest dose of Lionfish protein isolate. Indeed, the inhibition effect on HeLa cells can be enhanced because SDS-PAGE results showed that there are several proteins with different effects on tumor cells contained in the protein isolate of Lionfish venom. Thus, eliminating these proteins might increase the inhibition effects of Lionfish venom protein in tumor cells. These results suggest that Lionfish has an antitumor effect, but need further purification step and more evaluation of the protein in Lionfish venom extract that can enhance the antitumor effect. Keywords: Antitumor, Lionfish, Protein isolation
INTRODUCTION The utilization of venom as an alternative therapy has received much attention in recent years due to its therapeutic effects in several chronic diseases like tumors or cancer, which offer some economic and ecological benefits. Lionfish (Pterois volitans) is a native fish species in the Indo-Pacific Ocean. Lionfish (Pterois volitans) is one of the invasive species in the oceans. The high reproduction and distribution rates of Lionfish cause the widespread of Lionfish invasion. Twenty years from the first time the Lionfish was discovered, the Lionfish invasion area has spread to most of western Atlantic, Florida, Bahamas, Cuba, Dominican Republic, Puerto Rico, most of the Lesser Antilles, Cayman Islands, Jamaica, Colombia, Costa Rica, Belize, Venezuela, and Mexico [1]. However, it has been found that the invasion of P. volitans has caused some negative impacts on marine ecosystems. The growth of P. volitans population has reduced the population of local fish species, thus affecting the economic conditions of the invaded area. The invasion of P. volitans in the US has caused financial losses of up to
The 4th Biomedical Engineering’s Recent Progress in Biomaterials, Drugs Development, Health, and Medical Devices AIP Conf. Proc. 2193, 030009-1–030009-8; https://doi.org/10.1063/1.5139346 Published by AIP Publishing. 978-0-7354-1944-5/$30.00
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120 billion USD [2]. In the Carribean islands, uncontrolled P. volitans population has also created the damage of coral reefs and marine ecosystems. The revenues from the tourism industry in the Carribean islands decreased by up to 2-5% with an estimated loss of 100 to 300 million USD [3]. One way to reduce the P. volitans population level is by using P. volitans to become something that can be useful. P. volitans produce toxins that have the cardiovascular, neuromuscular and cytolytic effect (causing cell death) associated with the presence of some toxic proteins and other active components such as acetylcholine or noradrenaline and toxin pore-forming [3]. Based on recent studies, the cytolytic effects of the cytotoxic activity of Lionfish (Pterois volitans) from Indian waters, were able to reduce the growth of tumor cells (Erlich's Ascites Carcinoma cells). Protein with a molecular weight 7.6 kDa was isolated from crude venom and tested to tumor cells, and the results showed that there is antitumor activity [4]. The reduction of tumor cells was caused by cytotoxic activity found in P. volitans venom. The toxins play an indirect role in destroying the microenvironment where the tumor cells grow and induce an apoptosis mechanism, which is causing DNA fragmentation by activating the caspases enzyme in tumor cells [5]. Apoptosis activity that occurs in cancer cells was created because P. volitans venom activates caspase-8, which can activate procaspase-3 to activate caspase-3 [6]. However, although the effect of P. volitans venom on tumor cells line was demonstrated over twelve years ago, little attention has been paid to the process of isolating the antitumor protein. One of the conventional protein isolation methods is precipitating protein using ammonium sulfate. Lionfish protein isolation using ammonium sulfate has been done before by Savitri, et al. to observe the phospholipase A2 (PLA2) activity on Lionfish venom [7]. However, the effects of ammonium sulfate concentration added for isolating the antitumor protein is never explained. The present paper explains the effects of ammonium sulfate concentration on the protein of P. volitans venom isolation on an antitumor test and evaluates the activity and the content of its antitumor protein.
MATERIAL AND METHODS Pterois volitans The specimens of Pterois volitans were obtained from around the Indonesian oceans. Lionfish was killed (by cooling), and the venomous spines were removed and washed with 0.1 M phosphate buffer (pH 7.0) and 0.01 M CaCl 2. P. volitans was chosen for this experiment because it is one of the most invasive species and its a common in Indonesia.
HeLa Cell Culture The tumor cells line that was chosen for this research is HeLa cells. HeLa cells culture was obtained from Indonesia International Institute for Life Sciences (I3L, Indonesia). HeLa cells that used for this research were harvested when it reached its log phase. Venom isolation on an antitumor test and evaluates the activity and the content of its antitumor protein.
Extracting The Crude Venom P. volitans spines were extracted using the methods described by [7]. Briefly, the spines (taken from upper dorsal, bottom and back fins) were measured and soaked in 0.1 M phosphate buffer pH 7.0 (which have mixed by CaCl 2 solution with the ratio between phosphate buffer and CaCl2 added is 1:10). The ratio between the weight of spines and the phosphate buffer solution added was 1:2. The spines that have soaked in phosphate buffer solution were sonicated for 16 minutes (with frequency 20 kHz). The extract was centrifuged at 4,500 rpm for 30 minutes. The supernatant (crude venom) was obtained from this process.
Isolating Protein Using Ammonium Sulfate Precipitation The ammonium sulfate was added to crude venom that obtained from the extraction process. This process was done to obtain the protein precipitate from crude venom. The crude venom was aliquoted into five individual volumes (5 mL). 20%, 40%, 60%, and 80% saturated ammonium sulfate was added to each 5 mL of crude venom (the amount of ammonium sulfate that should be added was referred to ammonium sulfate saturation table by [8] and then stirred
030009-2 with vortex or stirrer for 30 minutes. The protein precipitate was separated by removing the supernatant after the centrifugation process at 4,500 rpm for 30 minutes. The amount of saturated ammonium sulfate added to obtain protein precipitate become the independent variables of this experiment
Protein Concentration Measurement The protein concentration of each sample was measured by the Lowry test. The measurement of protein concentration was used to make sure the precipitate that obtained in the previous step is protein. The protein concentration was obtained from five samples. These samples were crude venom (CV), protein precipitate from the addition of 20%, 40%, 60% and 80% saturated ammonium sulfate (AS20%, AS40%, AS60% and AS80%). All of five samples were added by 5 mL biuret reagent and incubated for 10 minutes. 0.5 mL of Folin-Ciocalteu reagent was added to the samples that have incubated. The absorbance of the samples was measured with wavelength 750 nm, and then the data were plotted to Lowry’s curves standard to obtain the protein concentration of the samples.
Measurement of LC50 using Brine Shrimp Lethality Test The procedures of LC50 measurement were referred to [9]. The measurement of LC50 was done to check the activity of a bioactive compound that contains protein precipitate. Briefly, each of the samples (AS20%, AS40%, AS60%, and AS80%) were diluted into several concentrations (10, 100, 500, and 1000 ppm) by using seawater. Samples that have been diluted into several concentrations were tested to ten A. salina larvas which have hatched for 2x24 h. A. salina larvas were obtained and hatched by Biofarmaka Laboratory, Institut Pertanian Bogor (West Java, Indonesia). The data of the amount of death larva collected after the samples incubate for 24 hours. To obtain the value of LC50, the amount of death larva was calculated using probit analysis regression methods [10], and the activity of the bioactive compound was evaluated using Clarkson’s toxicity index [11].
Identification of Protein Molecular Weight using SDS-PAGE Five samples (CV, AS20%, AS40%, AS60%, and AS80%) were tested using SDS-PAGE to identify the molecular weight of protein precipitate samples. Each of the samples was heated at 100°C for 45 seconds to break the protein binding. 4μL phosphate-buffered saline and 5μL sample buffer was added to 5μL samples (CV, AS20%, AS40%, AS60%, and AS80%) that have heated first. The samples were run into the SDS-PAGE with power 150 V for 1 hour. Running buffer was released, and the gel was taken away from the SDS-PAGE set. Then, the staining process using the coomassie blue and the destaining process using methanol were done. The molecular weight of the protein was determined by comparing the sample gel and the marker protein.
Microculture Tetrazolium Salt Assay (MTT Assay) MTT assay used for evaluating the antitumor effect of samples. The procedures used for this experiment were referred to as CellTiter 96° Nonradioactive Cell Proliferation Assay Instruction. Promega G400 used as a reagent in this assay. Each of samples (CV, AS20%, AS40%, AS60%, and AS80%) was diluted into several concentrations (2, 250, 500 and 2500 ppm) using DMEM, PenStrep and FBS 0.5% solution. 2 x 10 4 of log-phase HeLa cells were seeded into each well and incubated a day prior to treatment with lionfish venom samples. After the cells were treated with different concentrations of the samples, they were incubated for another 20 hours. MTT dye solution was then added into each well and incubated for 4 hours before the stop solution was added. One hour later, the absorbance values at 570nm of each well were then recorded. All treatments were done in duplicate wells. Cell viability was calculated by dividing the absorbance value of each sample concentrations with the absorbance value of untreated HeLa cells.
RESULTS Protein Concentration The protein concentration raised along with the increase of the amount of saturated ammonium sulfate added. The protein precipitate sample obtained from protein isolation using 80% saturated ammonium sulfate (AS80%) resulted in the highest protein concentration.
FIGURE 1. The graphic showed the effects of protein concentration obtained vs. the variation of the amount of saturated ammonium sulfate added. The values attached to the graph were the protein concentration value obtained.
However, the lowest protein concentration was obtained by the protein precipitate sample that was isolated using 20% saturated ammonium sulfate (AS20%). The higher addition of saturated ammonium sulfate added caused the increase of protein concentration slightly. Compared to crude venom sample, samples that isolated using high amount of ammonium sulfate gave a higher result of protein concentration (Figure 1), it’s because the crude venom samples have not gone through the purification process before, so there are still many impurities that inhibit the reaction between the Lowry reagent and protein in crude venom sample.
Lethal Concentration 50 (LC50) LC50 values obtained from Brine Shrimp Lethality Test (BSLT) data. All of the samples (AS20%, AS40%, AS60%, and AS80% that tested to a salina larvas showed the toxicity activity. The toxicity activity indicated that the bioactive compound in the sample was active and the protein isolation process using ammonium sulfate precipitation succeed in precipitate protein, which there was a bioactive compound. TABLE 1. The LC50 value and toxicity level of P. volitans protein venom sample Protein Sample LC50 (ppm) Toxicity Level AS20% 796.66 Less Toxic AS40% 300.01 Moderate Toxic AS60% 196.28 Moderate Toxic AS80% 101.93 Moderate Toxic
The LC50 value that showed on the Table 1 was calculated using probit analysis regression method. The toxicity level of the sample was evaluated using the Clarkson toxicity index [11]. Based on LC50 results, all of the samples
030009-4 categorized as toxic by Clarkson toxicity index. The toxicity rate increased moderately along with the rise of the amount of saturated ammonium sulfate added to crude venom. The lower LC50 value indicated the higher level of toxicity and the higher activity of the bioactive compound. AS80% sample resulted in the highest toxicity level, which categorizes ad moderate toxic. On the other hand, the AS20% sample was the sample with the lowest toxicity level.
Protein molecular weight P. volitans protein venom sample that was tested (CV, AS20%, AS40%, AS60%, and AS80%) produced protein bands on an SDS-PAGE gel. The thickest protein band generated by AS40%, AS60%, and AS80% sample. The thickest protein band obtained showed the more protein contained in the sample. A protein band of AS20% and CV samples were not clear because the protein content in the sample was low (not as high as AS40%, AS60%, and AS80% sample). Therefore the molecular protein weight could not be determined.
FIGURE 2. SDS-PAGE gel result contained protein bands that were separated based on its molecular weight. (A) is CV sample, (B) AS20%, (C) AS40%, (D) AS60% and (E) AS80%. Protein molecular weight was determined by comparing the protein band that was formed with a protein marker. TABLE 2. Protein identification results based on its molecular weight Protein Molecular Weight Name, function