Most of our venoms have been purified and characterized. In some case other medical investigators would like a certain purified fraction. To provide this service, venoms will be purified and characterized. High performance liquid chromatography (HPLC) for size exclusion, ion exchange, and reverse phase will be used. HPLC has provided a very convenient and rapid method for toxin isolation. This method is commonly used at the NNTRC and has proven to be successful (Khunsap et al., 2011; Teklemariam et al., 2011; Suntravat et al., 2010; Da Silva et al., 2009; Salazar et al., 2009; Sánchez et al.,2009; 2006; 2005; Galán et al., 2008; Pineda et al., 2008; Girón et al., 2007). Molecules that could have medical applications such as disintegrins, C-type lectins, PLA2s, snake venom metalloproteinases and serine proteases will be isolated from A. piscivorus leucostoma, C. adamanteus, C. viridis viridis and C. horridus.
Method for Purification
Chromatographic steps for purifying molecules from snake venoms depend on their physical and chemical characteristics. These methods are routinely used at the NNTRC. The method for purification of disintegrins consists of a combination of three chromatographic steps: reverse phase C18 (Vydac), size exclusion, and anion exchange chromatography (Sánchez et al., 2009; 2006; 2005; Galán et al., 2008).
Two hundred microliters of lyophilized crude venom in the appropriate buffer at the concentration of 25 mg/mL will be injected into a Grace Vydac Reverse Phase C18 (4.6 x 250 mm) column. Fractions will be eluted using a 0.1% trifluoroacetic acid (TFA), and 80% Acetonitrile (ACN) in 0.1%TFA gradient over 60 min with a flow rate of 1 mL/min. Proteins will be detected at 280 nm. The fractions will be tested for inhibition of ADP-induced platelet aggregation
Size Exclusion Chromatography
Fractions collected with a reverse-phase C18 chromatography inhibiting platelet aggregation will be further fractionated by size exclusion chromatography. Two hundred microliters of pooled fractions will be injected into a Waters™ ProteinPak 60 column (7.8 x 300 mm) on a Waters™High Performance Liquid Chromatography system. The buffer used will be 0.02 M Sodium phosphate at pH 6.2, over 60 min with a flow rate of 0.5 mL/min. Proteins will be detected at 280 nm. The fractions will be tested for inhibition of ADP-induced platelet aggregation.
Anion Exchange Chromatography
Fractions collected using size exclusion chromatography inhibiting platelet aggregation will be further fractionated by anion exchage chromatography. Two hundred microliters of pooled fractions will be applied into a Waters PROTEIN-PAKTM 5PW (7.5 x 75 mm) column on a Waters HPLC. The buffer used will be 0.02 M Tris-HCl, pH 8.0, with the eluting buffer containing 0.5 M NaCl, over 60 min with the flow rate of 1 mL/min. Proteins will be detected at 280 nm. The fractions will be tested for inhibition of ADP-induced platelet aggregation and other disintegrin assays including inhibition of cell migration, cell adhesion, metastasis, and angiogenesis.
The most effective way of determining the mass of proteins and peptides is mass spectrometry. This is a common method used at the NNTRC (Khunsap et al., 2011; Sánchez et al., 2010; Salazar et al., 2009; Galán et al., 2008).
Method for Mass Analysis
Protein samples will be dried in an Eppendorf speedvac for 30 min at 30°C, resuspended in 10 L of 0.1% TFA/50% ACN, and desalted using C18 Zip Tip (Millipore ZTC18S096). Five hundred nanoliters of -cyano-4-hydroxycinnamic acid (Bruker Daltonics) will be spotted on a MTP AnchorChip target plate 600/384 TF (Bruker Daltonics) and 0.5 μL of sample will be added onto the matrix. MALDI-TOF analysis will be performed using a Bruker AUTOFLEX II-TOF (Bruker Daltonics) in positive mode. A Peptide Standard 2 (Bruker Daltonics) including insulin-5735.89, ubiquitin-8573.6, cytochrom-12370.83, myoglobin-16948.86, cytochrome-6183.08, and myoglobin-8459.67 will be performed in a reflectron mode.
Use of Human Blood
This research project will require human blood to screen for disintegrins in snake venoms that block human platelet aggregation. Human blood will also be used in blood agar plates for the detection of phospholipase. The blood will only be used as a reagent and no human records will be kept. All materials used for blood collection will be sterile and single-use.
Before and after purification, biological assays will be used to screen venom and venom fractions for hemorrhagic, proteolytic, fibrinolytic, fibrinogenolytic, and gelatinase activities. In addition, disintegrin assays including those that block metastasis, angiogenesis, and platelet aggregation will be used. Other assays will be used to better define the biological functions of peptides that prevent metastasis. The platelet aggregation assays and cell binding assays are routinely conducted at the NNTRC. Molecules that show potential for tumor reduction will be screened in BALB/c mice.
Method for Hemorrhagic Assay
A hemorrhagic assay will be performed to determine which venoms and fractions cause bleeding. We routinely use a modified method described by Sánchez et al. (2011) to determine the minimal hemorrhagic dose (MHD) for the crude venoms. A series of dilutions will be made for each venom sample, of which 0.1 mL of each dilution will be injected subcutaneously into the backs of BALB/c mice (n=8) to test for activity. After 24 hr, the mice will be sacrificed. The hemorrhagic spots will be measured (mm) and the MHD determined. The MHD is defined as the amount of venom protein, which causes a 10 mm hemorrhagic spot. After the results are obtained the mice will be fed to the snakes.
This assay has been used routinely to confirm the purification of fibrinolytic molecules and to screen all venom for fibrinolytic activities (Sánchez et al., 2005).
Method for Fibrinolytic Activity
Three hundred microliters of fibrinogen solution (9.4 mg/mL) and 10 µL of thrombin solution (38.5 U/mL) will be added to each well of a 24-well plate, which will be then shaken gently. The solution in the plate will be allowed to clot at room temperature. The plate will be then incubated for 3 hr at 37˚C. Ten microliters of one venom fraction is added per well and incubated overnight at 37˚C. Seven hundred microliters of 10% trichloroacetic acid (TCA) will be placed in each well, then poured off after 10 min. The specific fibrinolytic activity is calculated by dividing the diameter cleared (mm) by the amount of protein sample (µg) placed in each well.
Hide powder Azure Assay
Snake venom contains a mixture of thousands of bioactive toxins, many of which are components with enzymatic activity. The Hide Powder azure assay is an inexpensive, fast, and easy technique to identify proteases.
Method for Hide Powder Azure Assay
A modified method of Rinderknecht et al. (1968) will be used to test proteolytic activity. Hide powder azure will be ground with a mortar and pestle to obtain uniform particles before adding diluent. Eight milligrams of finely ground hide powder azure will be mixed with 2 mL of 0.02 M Tris-HCl buffer, pH 8.0, and 100 µL of each venom fraction will be then added to the vial. Each vial will be incubated for 1 hr at 37˚C and the absorbance measured at 595 nm. The specific activity will be calculated by dividing the absorbance by the amount of protein used (mg).
The gelatinase assay is an inexpensive, simple and rapid proteolytic assay that will be used in screening venoms, purified fractions, and cDNA expressed products. This method will be helpful in identifying serine protease and metalloproteinase enzymes when they are used in the presence of specific inhibitors of proteases.
Method for Gelatinase Assay
A method modified from Huang and Pérez (1980) will be used to test gelatinase activity of the venom fractions. Twenty microliters of each venom fraction will be placed in separate locations on a Kodak X-OMAT scientific imaging film with gelatin coating. Hydrolysis of gelatin on the X-ray film will be determined by washing the film with tap water after a 4-hr incubation at 37˚C in a moist incubator.
The Sonoclot® Analyzer is an instrument intended for general hemostasis monitoring. This instrument can measure the onset time of coagulation, the rate of fibrin clot formation, platelet function, and fibrinolysis
Method for Sonoclot® Analyzer Profiles
We have published data that indicates that this instrument is useful in identifying proteins with procoagulant, anticoagulant, and anti-platelet activities (Sánchez et al., 2010; Suntravat et al., 2010). The platelet function can be used to screen venoms for disintegrins. A glass-bed-activaded test (gbACT + KIT obtained from Sienco, Inc) will be used to monitor the effect of venom fractions on the clotting process. Ten percent citrated human blood will be incubated to 37˚C at least 5 min prior to use. A total of 13 µL of 0.25 M CaCl2 will be added to one side of a gbACT+KIT cuvette and then 10 µL of disintegrins at the same molar concentrations or 0.85% saline (negative control) will be added to the opposite side of the cuvette. Recombinant disisntegrin, r-mojastin 1, will be used as a positive control (Sánchez et al. 2010). After both solutions are added, 360 µL of citrated blood will be added to the cuvette and the analyzer will be activated. Data will be collected by a Signature Viewer™ program v. 3.1 on an iMac computer containing Mac OS X software.
The aggregometer is a useful instrument for determining anti-platelet aggregation in whole blood, but may also indicate the presence of disintegrins, which prevent metastasis. Positive fractions will be confirmed by cellular adhesion assays.
Method for the Aggregometer Assay
A Chronolog™ Whole Blood Lumi-Aggregometer will be used to monitor the inhibition of platelet aggregation by measuring impedance. Four hundred and fifty microliters of 10% citrated human blood will be incubated at 37˚C at least 5 min prior to use with equal amounts of 0.15 M saline solution. Ten microliters of the venom fraction will be incubated with the blood sample for 2 min at 37ºC. Platelet aggregation will be initiated by 10 µL of ADP (10 µM final concentration), and percentage of impedance reflecting percentage of aggregation will be measured. The percent inhibition of platelet aggregation will be calculated by the following equation: [(C-E/C] x100, where C is the units of platelet aggregation (ohms) for the control, and E is the unit of platelet aggregation (ohms) for the experimental fraction. The negative control will consist of whole blood incubated with 10 µL of 0.15 M saline solution. The recombinant disisntegrin (r-mojastin 1) will be used as a positive control (Sánchez et al. 2010). The extent of the inhibition of platelet aggregation will be assessed by comparison with the maximal aggregation induced by the control dose of agonist (10 µM ADP) and expressed as 50% inhibition concentration (IC50) as determined from a dose-response curve generated from the various antagonist concentrations.
Factor X Activator Assay
This is a simple test routinely used to measure Factor X activator activity.
Method for Factor X Activator Activity
Factor X activator activity in venom or venom fractions will be tested using factor Xa-specific chromogenic substrate S-2765 as described by Suntravat et al. (2011; 2010). Twenty-five microliters of the pooled normal human citrated plasma (50 mg/mL) dissolved in Tris-EDTA buffer will be incubated at 37°C for 3 min. Then, 25 μL of chromogenic substrate S-2765 will be added and mixed within 30 sec. The 25 μL of venom or venom fractions premixed with 0.1 M CaCl2 in equivalent quantities will be added and incubated at 37°C for 3 min. The reaction will be stopped by 20% acetic acid. The samples will be monitored at 405 nm. Each test was performed in duplicate.
cDNA Library Construction Method
We will take advantage of existing snake resources at the NNTRC to construct cDNA libraries using venom glands. It estimated that two cDNA libraries could be constructed per year. The first will be from the venom glands of C. horridus snake. Snake glands will be dissected from euthanized snakes, and immediately frozen in liquid nitrogen. The whole procedure of cDNA library construction was detailed in our publication (Jia et al., 2008). Approximately 20 µg of the gland will be used to extract total RNA by using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). A directional cDNA library using 0.5 µg of total RNA will be constructed by using a Creator SMART cDNA Library Construction Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) based on the manufacturer’s instructions with minor modifications. First, the total RNA (0.5 µg) from the venom glands will be subjected to reverse- transcription and cDNAs will be synthesized using PowerScript Reverse Transcriptase (Clontech) and the CDSIII/3’ PCR primer (Clontech) for 1 hr at 42°C. Second, double stranded cDNA (ds cDNA) synthesis will be performed on an iCycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by long-distance (LD) polymerase chain reaction (PCR). Finally, the digested ds cDNA will be fractionated using a CHROMA SPIN-400 column (Clontech). Samples of 3 µL of each fraction will be electrophoresed on a 1.1% agarose/EtBr gel to determine the peak fractions by visualizing the intensity of the bands under an UV light. The fractions will be pooled as large-, medium- and small-sized cDNA, concentrated and separately ligated into pDNR-LIB vector (Clontech) for overnight at 16°C. The resulting ligation reactions will be transformed into one shot Electrocomp GeneHogs E.coli (Invitrogen Corporation, Carlsbad, CA, USA) separately. The three libraries (large-, medium- and small-sized) will be mixed for the unique snake venom cDNA library. All of the procedures are routinely used at the NNTRC (Jia et al., 2008; Sánchez et al., 2010; Suntravat et al., 2012 unpublished). cDNA clones will be sent to DNA Facility of the Iowa State University for sequencing using the Applied Biosystems 3730xl DNA Analyzer.
Subcloning, Expression and Purification
PCR will be performed to amplify the full-length gene using cDNA clones as the PCR templates. PCR products will be extracted and precipitated using ethanol in -80°C for 1 hr. The pellet will be washed in 70% ethanol, dried, dissolved in H2O and subcloned into a pGEX-4T-1(GST) vector (Amersham Biosciences). GST-gene will be transformed into XL blue competent cells. Plasmid DNA will be extracted using miniprep kit and sequenced for confirmation of in-frame with GST vector (Jia et al., 2009; Jia and Perez, 2009).
The confirmed plasmid, GST-gene will be transformed into the E.coli strain BL21 star to give strain BL21/GST-gene. The recombinant strain will first be cultured in shake flasks containing Luria-Bertani (LB) medium overnight. After seeding the overnight culture into fresh LB medium, the growth of culture cells will be maintained at 37°C and monitored turbidimetrically at 600 nm along the time course. Upon reaching OD600 of 0.5, the culture will be induced with 100 µM Isopropyl-D-thiogalactoside (IPTG) for 3 hr for recombinant protein induction. After incubation, cells will be harvested by centrifugation and suspended in 1 x phosphate buffered saline (PBS) solution. Cells will be lysed on ice with a Branson Sonifier 450 (Branson, Danbury, CT) with the output control setting at 1, a duty cycle setting of constant, and 6 sonication pulses of 30 sec per pulse. The lysate will be centrifuged for 15 min at 10,000g. The supernatant will be affinity purified with Glutathione-Sepharose 4B beads in Econo-Column (BIO-RAD) on an ECONO PUMP (BIO-RAD) at the flow rate of 0.3 mL/min. The column will then be washed with 30 mL ice-cold 1 x PBS buffer at the flow rate of 0.3 mL/min. The GST-gene fusion protein will be eluted from beads by 10 mM Glutathione Elute Buffer (GEB) or the recombinant proteins will be cleaved from the GST-protein using thrombin protease in the column for 2 hr at room temperature, and eluted with 1 x PBS buffer. The eluted protein will be passed through a HiTrap Benzamidine FF column (GE Healthcare) to remove thrombin protease. The purity of the recombinant protein will be determined by SDS electrophoresis using NuPAGE 4-12% Gel (Invitrogen).
Inhibition of Cellular Adhesion Assay
The cellular adhesion assay will be used in the resource and research components of the proposed research. The cellular adhesion assay is an efficient and convenient way to assay for disintegrins that inhibit the binding of a wide variety of cell lines to different ligands, and it is, therefore, necessary to test a wide array of cancer cell lines containing different integrins. (Lucena et al., 2012; Lucena et al., 2011; Sánchez et al., 2009).
Method for Inhibition of Cellular Adhesion Assay
A total of 12 cell lines will be used in this procedure (human urinary bladder carcinoma cell line (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorectal adenocarcinoma (CaCo-2), human breast adenocarcinoma (MDA-MB-231), human lung bronchus carcinoma (ChaGo-k-1), murine mammary breast carcinoma cells (66.3p), murine skin melanoma (B16F10), and human pancreatic cancer cell lines BxPC3, Panc1, Capan1, and AsPC1. These are all cell lines that have been purchased and stored at the NNTRC. Venom fractions will be assessed for their specific binding by a cell adhesion assay as described by Lucena et al. (2011). Triplicate wells in a 96-well plate will be coated with matrix ligands such as fibronectin, vitronectin, collagen, or laminins at 10 µg/mL in 0.01M PBS, pH 7.4, and incubated overnight at 4°C. The plate will be blocked with 0.2 mL of PBS in 5% Bovine serum albumin (BSA) and incubated at 37°C for 1 hr. Cells will be harvested, counted, and re-suspended in minimum essential medium (MEM) medium containing 1% BSA at 5 x 105 cells/mL. Disintegrins will be added to the cell suspension at various concentrations and allowed to incubate at 37˚C for 1 hr. The blocking solution will be aspirated, and the cell/disintegrin suspensions (0.2 mL) will be added to the wells coated with matrix protein and incubated at 37°C for 1 hr. The solution will be aspirated and washed three times with PBS-5% BSA by filling and aspirating. A total of 0.2 mL of MEM medium in 1% BSA containing 3-[4,5-Dimethylthiazol-2-yl] 2,5-diphenltetrazolium bromide (MTT) (5:1 vol/vol) will be added to the wells containing cells and incubated at 37°C for 2 hr. A total of 0.1 mL of Dimethyl sulfoxide (DMSO) will be added to the wells to lyse the cells. The plate will be shaken gently and the absorbance will be read at 570 nm using a Beckman Coulter model AD 340 reader.
Cell Migration Assay
The cell migration assay will be used in the resource and research components of the proposed research. Migration and invasion of tumor cells to the vascular extracellular matrix are a major part of metastasis. We will evaluate the effect of isolated and cloned peptides on cell migration using different tumor cell lines (Lucena et al., 2012; Lucena et al., 2011; Galán et al., 2008).
Method for Cell Migration Assay
Tumor cell migration will be measured after scraping cells from the bottom of the well as described by Galán et al., (2008). This technique is routinely used at the NNTRC. Commercial echistatin at 2.7 µM, a disintegrin that blocks migration of tumor cells, will be used as a positive control. The positive control prevents tumor cell migration. The negative control consisted of tumoral cells incubated with PBS, which allows cell migration to occur. Cells will be incubated in a CO2 chamber and only removed from the incubator for microscopy images at times 0, 3, 6, 12, and 24 hr after disintegrin incubation. The percent of closure will be calculated by the following equation: % Closure: [(C-E)/C] x 100, where C is the units of distance of cell edge (mm) at zero time for the control, and E is the distance from the cell edge (mm) at the final incubation time for the disintegrin. The cell migration results will be expressed as the mean ± standard deviation (n=3), and analyzed using the one way-Anova test followed by Newman-Keuls Multiple Comparison Test, using the software program Graph Pad Prism.
Matrigel Plug Angiogenesis Assay
The in vivo angiogensis assay will be used in the resource and research components of the proposed research. Tumor cell invasion alone is not sufficient to produce metastases. In order to survive and metastasize, the cancer cells need their own supplies of nutrients and oxygen; this is achieved by the formation of tumor angiogenesis. The importance of angiogenesis to the pathogenesis of cancer makes it necessary to determine the anti-angiogenic activity of the disintegrins using an in vivo angiogenesis assay (Lucena el at., 2012 unpublished).
Method for Matrigel Plug Angiogenesis Assay
The matrigel plug assay will be performed with a modified method of Yeh et al., 2001. An aliquot (0.5 mL) of matrigel, supplemented with basic fibroblast growth factor (bFGF) (500 ng/mL) in the presence or absence of disintegrins, will be injected subcutaneously into the dorsal region of BALB/c mice (weight 18-20 g). After 15 days, matrigel plugs will be removed, dissected free from adherent tissue, weighed, and homogenized for hemoglobin quantification following the colorimentric method described by Higuchi et al (2011). Matrigel plug will be homogenized in 2.0 mL Drabkin reagent (Sigma-Aldrich, St Louis, MO) and centrifuged at 10,000g for 15 min. The supernatants will be filtered through a 0.22 µm filter (Millipore, Bedfor, MA) and the hemoglobin in the samples will be determined spectrophotometrically at 540 nm. The amount of hemoglobin will be calculated with a known standard. The results will be expressed as µg Hb mg-1 of wet tissue.
In vivo Inhibition of Lung Colonization of Tumoral Cells Assay
The lung colonization assay will be used in the research component of the proposed research. An important test to verify the effectiveness of the disintegrins as anti-cancer agents is the inhibition of lung tumor colonization in vivo. This test will be used to determine if disintegrins can be used to block lung tumor metastasis in animal models and will only be done with disisntegrins that show positive activity in vitro assays (Lucena el at., 2011; 2012).
Method for In vivo Inhibition of Lung Colonization of Tumoral Cells Assay
Lung colonization of tumoral cells will be performed as described by Lucena et al. 2011. Murine melanoma cells (B16F10) and/or murine mammary breast carcinoma cells (66.3p), (5.0 x106 cells/mL) will be suspended in Dulbecco’s modified Eagle’s medium or MEM without FBS, respectively, in the presence or absence of disintegrins at 1000 µg/kg per mouse and incubated 1 hr at 37ºC. Controls will consist of mice injected with murine tumoral cells in medium without FBS and mice injected with medium alone. We will use eight mice in each group. Two hundred microliters of cells/disintegrins mixture will be injected intravenously (i.v) in the lateral tail vein of BALB/c mice (18-20 g). Mice will be sacrificed 19 days post injection, and lungs will be examined for the presence of tumors. Lungs will be visualized with a 4 x stereomicroscope. The tumors will be counted and statistically analyzed. A two tailed t-test followed by a Mann Whitney test will be used to determine the significance of disintegrins and the control in inhibiting the number of tumors in vivo.
Inhibition of Disintegrins in Pancreatic Cancer Cell Metastasis Assay
The nude mice assay will be used in the research component of the proposed research. Nude mice are one of the most widely used models to study human cancer. To identify potential of disintegrins as anti-cancer agents, we will use nude mice, which are immunodeficient mice and do not reject human cells for establishing a mouse peritoneal pancreatic cancer dissemination model. This test will be used to examine the response of human cancer, not mouse cancer, to disintegrins.
Method for Inhibition of Disintegrins in Pancreatic Cancer Cell Metastasis Assay
Four-pancreatic cell lines that have strongly invaded an artificial basement membrane will be used to study the anti-metastatic activity in nude mice. The protocol will be performed by a modification of the procedure of Fujiwara et al. (2011). The cells will be mixed with disintegrins at various concentrations (g/kg) and incubated for 1 h at 37°C. The mixture will be injected intraperitoneally (i.p.) into BALB/c nude mice (weight 18-20 g; n=8 each group). Mice will be sacrificed 24 days after the implantation of cells to evaluate the anti-metastasis activity of the disintegrins. The number of peritoneal nodules will be counted. The number and weight of nodules more than 5 mm in diameter will be measured. The positive control group will injected with cells incubated with PBS. The negative control group will be injected with PBS alone. All experiments and procedures will be approved by the IACUC and will be conducted in accordance with the National Institute of Health Animal Care Guidelines.
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