This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included.
Cancer is the third leading cause of death in the United Arab Emirates (UAE), after cardiovascular diseases and accidents. In the UAE, colorectal cancer (CRC) is the first and fourth most common cancer in males and females, respectively. Several treatment modalities have been employed for cancer treatment, such as surgery, radiotherapy, chemotherapy, hormone replacement therapy, and immunotherapy. These treatment modalities often elicit adverse effects on normal cells, causing toxic side effects. To circumvent these toxicities, there has been an increased impetus towards the identification of alternate treatment strategies. Animal venoms are rich sources of pharmacologically active polypeptides and proteins.
In this proof-of-concept study, we will apply a high-throughput venomics strategy to identify and characterize anticancer bioactive peptides (BAPs) from 20 different animal venoms, specifically targeting CRC. We chose to focus on CRC because it is one of the foremost health issues in the UAE.
In the initial study, we will screen 2500 different peptides derived from 20 different animal venoms for anticancer activity specifically directed against 3 CRC cell lines and two control cell lines employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay for cytotoxicity. Of the 20 venoms, 3 that exhibit specific and potent anticancer activity directed against the 3 CRC cell lines will be selected; and from these 3 venoms, the specific peptides with anti-CRC activity will be isolated and characterized.
This study is at the protocol development stage only, and as such, no results are available. However, we have initiated the groundwork required to disseminate the proposed study, which includes culturing of colorectal cancer cell lines and preparation of venom screens.
In summary, the proposed study will generate therapeutic leads to manage and treat one of the leading health issues in the UAE, namely, CRC.
PRR1-10.2196/31128
Cancer is the third leading cause of death in the United Arab Emirates (UAE) after cardiovascular diseases and accidents. In the UAE, colorectal cancer (CRC) is the first and fourth most common cancer in males and females, respectively [
Animal venoms are veritable gold mines of pharmacologically active polypeptides and proteins. In fact, proteins and peptides with anticancer properties have been identified and characterized from venoms of snakes, bees, scorpions, wasps, ants, spiders, and caterpillars [
In summary, animal venoms comprise a mix of bioactive peptides (BAPs), many of which exhibit anticancer activity against specific types of cancer mediated by one of the following four mechanisms: (1) induction of cell cycle arrest, growth inhibition, and apoptosis; (2) inhibition of angiogenesis; (3) inhibition of invasion and metastasis; and (4) blocking of specific transmembrane channels [
In this proof-of-concept study, we will apply a high-throughput venomics strategy to identify and characterize anticancer peptides from 20 different animal venoms, specifically targeting colorectal cancer. We chose to focus on colorectal cancer because it is one of the foremost health issues in the UAE.
The proof-of-concept study will address two specific aims.
In Aim 1, we will screen 2500 different peptides derived from 20 different animal venoms for anticancer activity specifically directed against 3 CRC cell lines and 2 control cell lines employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTT) colorimetric assay for cytotoxicity.
In Aim 2, 3 venoms of the 20 screened in Aim 1 that exhibit specific and potent anticancer activity directed against the 3 CRC cell lines will be selected; from these 3 venoms, the specific peptides with anti-CRC activity will be isolated and characterized.
The dissemination plan for the proposed research corresponding to Aim 1 and Aim 2 is shown in
Dissemination plan for the proposed research. CRC: colorectal cancer; HPLC: high-performance liquid chromatography; ROS: reactive oxygen species.
Aim 1 of the study is directed to the identification of three animal venoms which exhibit potent and specific cytotoxic activity on CRC cell lines (HCT116, Caco-2/TC-7, and HT29) and normal colon cell lines (CCD18 and NCM-460).
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
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 [
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 [
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 [
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).
Using a combination of different in vitro experiments, the anticancer properties of the BAPs will be characterized. The experimental protocols and the rationale for pursuing these experiments are indicated below.
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 [
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 [
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 [
RhoC is a metastatic protein that is found to be constitutively active in many types of cancers, including CRC [
Statistical analyses will be accomplished using the Student
This study is at the protocol development stage, and as such, no results are available. Experimental procedures in this study will be conducted in vitro and will not involve the use of animal models or samples, patient samples or data, or recruitment of human subjects. Therefore, research conducted as part of this study poses minimal risk and fits one of the exempt review categories as defined by institutional review board (IRB) regulations at Mohammed Bin Rashid University (MBRU). Further clarification and information can be obtained from the MBRU IRB at irb@mbru.ac.ae. We received funding for this study following review of our proposal. The funding ID for our research is MBRU-CM-RG2021-08. Additionally, we have initiated the groundwork for this study, which includes the purchase of CRC cell lines and preparation of the venom screen kit in collaboration with Latoxan.
Animal venoms are cocktails of pharmacologically active polypeptides and proteins. Therapeutic leads from venom have been successfully developed into drugs, such as the following examples.
This drug [
This peptidomimetic drug [
Ziconotide [
Our studies will identify novel BAPs with anti-CRC activity. These BAPs will act as therapeutic leads for the development of peptidomimetics; in the initial stages, we will apply a similar strategy to that depicted by Al-Amri et al [
Several studies are available in the literature in which the anticancer properties of animal venoms specifically targeting CRC have been investigated. However, these studies mostly focus on the anticancer properties of the whole venom rather than on the isolation and characterization of BAPs mediating the anticancer effect. For example, in the study by Al-Asmari et al [
7-aminoactinomycin D
bioactive peptide
bovine serum albumin
colorectal cancer
2,7-dichlorofluorescein diacetate
dimethyl sulfoxide
ethylenediaminetetraacetic acid
enzyme-linked immunosorbent assay
fluorenylmethoxycarbonyl
high-performance liquid chromatography
institutional review board
Mohammed Bin Rashid University
mass spectrometry
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
reactive oxygen species
United Arab Emirates
None declared.