This step of the project will be pursued in collaboration with Latoxan (Valence, France), a leading producer of animal venoms, with whom we have engaged in previous research collaborations. Briefly, a library of venom peptides will be created in 96-well plates. Venoms will be first cleared from molecules over 8500 Da (cytolytic enzymes). On the basis of high-performance liquid chromatography (HPLC), each venom will be split into 20 fractions. Each fraction is expected to contain 5 to 10 peptides at a 10 μmolar concentration in a volume of 100 μl. The strength of this strategy is that the separation of peptides using HPLC facilitates the identification of the active peptide once a hit (specific cytotoxic activity on CRC cell lines) is detected. Each 96-well plate will be filled with the fractions derived from 4 venoms, equaling 80 fractions and approximately 500 different peptides in total. Therefore, the entire screen contains 2500 venom-derived peptides separated in 400 lyophilized fractions from 20 different animal venoms (refer to Figure 1 for details of the species that will be included in the screen).

Human colorectal cancer cell lines (HCT116, Caco-2/TC-7, and HT29) and normal colon cell lines (CCD18Co and NCM-460) will be purchased from American Type Culture Collection. For cell culturing, RPMI 1640 (Sigma-Aldrich) medium will be used, supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin antibiotic cocktail (Sigma-Aldrich). The culture flasks (T25/T75) will be incubated in a 5% CO₂ humidified incubator at 37 ºC. After reaching 70% to 80% confluency, the cells will be split using 1% trypsin–ethylenediaminetetraacetic acid (EDTA) solution (Sigma-Aldrich).

High-throughput screening of 400 lyophilized venom fractions from 20 different animal species will be executed by measuring the cytotoxic activity of the fractions on CRC cell lines (HCT116, Caco-2/TC-7, and HCT29) and normal colon cell lines (CCD18 and NCM-460) using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Merck Millipore) using the methods depicted in [10,11]. First, the lyophilized fractions will be diluted in dimethyl sulfoxide (DMSO) for further experiments. Secondly, 3000 to 4000 cells/well will be grown in 96-well plates, and after 24 hours, the cells will be introduced to 2 μM concentrations of venom fractions in complete culture medium, maintaining a blank (no venom) as a negative control and 5-fluorouracil as a positive control. After 72 hours of incubation, the cells will be incubated with 10% MTT, followed by 3 hours of incubation in dark in a 5% CO₂ humidified 37 ºC incubator. The purple formazan formed will be dissolved in DMSO (Sigma-Aldrich), and the plates will be quantified by measuring the absorbance at 570 nm using a microplate reader (Hidex). The percent viability of the cells will be calculated as:

[(absorbance of experimental sample) – (absorbance of blank sample)] / [(absorbance of untreated sample) – (absorbance of blank sample)] × 100

IC₅₀ values and statistical significance will be determined using one-way analysis of variance.

For 3 animal venoms, fractions (containing peptides) that exhibit specific and potent cytotoxic activity on CRC cell lines will be selected and used for subsequent studies, as depicted in Aim 2. We aim to focus on peptides; therefore, during the preparation of the screen, we will eliminate the cytolytic enzymes in the venoms.

The 3 venoms identified in Aim 1 will be procured in bulk from Latoxan. BAPs with CRC cell line–specific cytotoxic activity will be purified from the venoms using a combination of size exclusion, ion exchange, and semipreparatory and analytical reverse-phase high-performance liquid chromatography on an AKTA Avant multidimensional chromatography system (GE Healthcare Systems) that is available in our laboratory. The purification strategy will be similar to that described by Banerjee et al [12,13]. At each step of purification, we will check for cytotoxic activity, employing the MTT assay to identify the fraction in which the BAP is present. The purified BAP fractions will be collected, lyophilized, and stored at −20 °C until further use.

Under Aim 1, once we have screened all the venom fractions, we will analyze the hits that we have achieved. For this part of the project, we will be focusing on the hits for which cytotoxic activity is specifically observed against HT-29 cells. We will select the 3 venoms corresponding to the peptides that exhibit the most potent cytotoxic activity specifically against HT-29 cells. The remaining information corresponding to the peptides that exhibit cytotoxic activity will be used to create a biobank of active peptides, which will be the focus of later studies.

Therefore, under Aim 2 of the study, we will focus on purifying BAPs from the 3 venoms that we have selected following the screening process. These BAPs will be purified to homogeneity using a combination of chromatographic techniques (as indicated above). These purified peptides will be further characterized to identify the molecular mechanism by which they mediate the anticancer activity. Because the characterization phase will require BAPs in significant quantities, we also will use fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis for bulk synthesis of the BAPs. In a later phase of the study, we will establish a bacterial expression system for the mass production of the BAPs identified and characterized in Aim 2 using the methodology depicted in [14].

The homogeneity and molecular weight of the BAPs will be determined by electrospray ionization mass spectrometry using a PerkinElmer Life Sciences API-300 liquid chromatography/tandem mass spectrometry (MS) system at the MS facility at SGS (Geneva, Switzerland). Typically, reverse-phase HPLC fractions will be directly used for analysis. The ion spray, orifice, and ring voltages will be standardized for analysis. Nitrogen will be used as a nebulizer and curtain gas. A LC-10AD pump (Shimadzu) will be used for solvent delivery (40% acetonitrile in 0.1% trifluoroacetic acid) at a flow rate of 50 μl/min. BioMultiview software (PerkinElmer Life Sciences) will be used to analyze and deconvolute the raw mass spectra.

The purified BAPs will be reduced and pyridylethylated using procedures described previously. Briefly, BAPs (0.5 mg) will be dissolved in 500 μl of denaturant buffer (6 m guanidine hydrochloride, 0.25 m Tris-HCl, and 1 mm EDTA [pH 8.5]). After the addition of 10 μl of β-mercaptoethanol, the mixture will be incubated under vacuum for 2 hours at 37 °C. 4-Vinylpyridine (50 μl) will be added to the mixture, and it will be maintained at room temperature for 2 hours. The pyridylethylated BAPs will be purified on a Jupiter C18 analytical column (4.6×250 mm) using a gradient of acetonitrile in 0.1% (volume/volume) trifluoroacetic acid at a flow rate of 0.5 ml/min.

N-terminal sequencing of the native and S-pyridylethylated BAPs will be performed by automated Edman degradation using a pulsed liquid-phase sequencer (PerkinElmer Life Sciences Model 494, Procise) with an online Model 785A phenylthiohydantoin derivative analyzer (Applied Biosystems).

Cell motility is a very important parameter in determining the survival and progression of cancer. Augmented cancer cell motility is the root cause of end-stage organ damage causing mortality [15]. The cell motility assay will be performed according to a procedure recommended by Rodriguez et al [16]. Briefly, CRC cells will be grown in a 60 mm Petri dish until they reach 80% confluency; then, a fine scratch will be made with the aid of a sterile pipette tip, and the scratch will be immediately photographed at hour 0. Next, cells will be supplemented with complete medium to allow them to grow. After 24 hours, the migration of the cells from the scratched area will be monitored microscopically. The width of the scratch at 0 and 24 hours will be measured, and the percentage of the gap covered by the cells will be calculated.

Cancer metastasis involves a slight elevation in the production of reactive oxygen species (ROS). Cancer cells appear to thrive on high levels of ROS compared to their normal counterparts, as cancer cells have developed augmented antioxidant systems [17]. ROS generation in CRC cells and normal cells after in the presence and absence of BAPs will be assessed using 2,7-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) using the manufacturer’s protocols. Briefly, CRC cells and normal cells will be plated in 6-well plates (0.3 million cells/well), and subsequently, subconfluent cells will be treated with BAPs for half an hour. Afterwards, the cells will be trypsinized and plated in new black 96-well plates, followed by incubation with 10 μM DCFH-DA for 4 hours at 37 °C. The fluorescence measurements will be performed in a Hidex microplate reader (Turku) at an excitation and emission wavelength of 485 nm and 538 nm, respectively.

The extent of apoptosis caused by BAP will be evaluated on a FACSAria III flow cytometer (BD Biosciences) using an annexin V and 7-aminoactinomycin D (7-AAD) apoptosis detection kit (BioLegend). Cells at a concentration of 0.3 M will be seeded in a 6-well plate and incubated in a 37 ºC 5% CO₂ humidified incubator. After 24 hours, the cells will be treated with benzopyrene and 5-fluorouracil (positive control) for 24 hours. Cells will be stained with annexin V and 7-AAD in the dark for 20 minutes according to the manufacturer’s protocols. FlowJo 10.7.1 software (BD Biosciences) will be used to analyze the data.

Elevated levels of vascular endothelial growth factor, IL-6, and IL-8 are proven biological markers indicating cancer progression [18,19]. Taking into consideration the proactive character of these cytokines (IL-8 and IL-6) in cancer, we will assess their expression in CRC cell lines and normal cell lines following BAP treatment using the method described by Duffy et al [18]. Briefly, 0.3 million cells will be seeded in complete culture medium containing 1% bovine serum albumin (BSA) and incubated in a 37 ºC 5% CO₂ humidified incubator for 6 hours. Purified BAPs will be introduced to the cells, followed by incubation for 24 hours. After 24 hours, the cell culture media will be collected and centrifuged to remove the cell debris. The adherent cells will be collected after trypsinization and will be counted to normalize the cytokine concentration. The assay will be executed as per the manufacturer’s guideline on enzyme-linked immunosorbent assay (ELISA) plates (ExtraGene) precoated with human IL-6 and IL-8. The colorimetric intensity will be measured on a microplate reader (Hidex) at 450 nm.